Methods for diagnosing bacterial vaginosis

ABSTRACT

Disclosed are methods for diagnosing Bacterial Vaginosis (BV). The disclosed methods generally include detecting select species of  Eggerthella  and/or  Prevotella , and optionally detecting select species of  Lactobacillus . Also disclosed are nucleic acid oligomers and related compositions for detection of a 16S rRNA or its encoding gene from select species of  Eggerthella, Prevotella , or  Lactobacillus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/543,944, filed Jul. 14, 2017, which is a national stage entry ofInternational Patent Application No. PCT/US2016/012589, filed Jan. 8,2016, and claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 62/101,907 filed on Jan. 9, 2015. The entire contents ofeach of the foregoing applications are incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Oct. 19, 2020, isnamed “GP324-03UT_SEQS_ST25b” and is 10,094 bytes in size

BACKGROUND

According to the National Health and Nutrition Examination Survey,nearly a third of women between the age of 14 and 49 have bacterialvaginosis (BV). See Allsworth and Peipert, Obstetrics and Gynecology109:114-120, 2007). BV is the most common cause of vaginal discharge anda reason many women seek medical attention. It is also associated withpreterm birth, low birth weight, pelvic inflammatory disease, anincrease in STD infections, including HIV, and a greater risk of passingHIV on to sex partners. See Srinivasan and Fredricks, InterdisciplinaryPerspectives on Infectious Diseases, Vol. 2008, Article ID 750479, 22pages, 2008). Women with bacterial vaginosis may have symptoms includinga malodorous vaginal discharge or irritation, however, as many as halfof the women with diagnosable BV have no clear symptoms (see Srivinvasanand Fredricks, supra).

Most researchers and the CDC consider bacterial vaginosis to be theresult of a disruption to the normal bacterial flora of the vagina.Unlike common infections, this dysbiosis is not the result of anindividual bacterial species. See CDC Factsheet, 2014(BV-Fact-Sheet-March-2014.pdf, from CDC website). A dysbiosis is adisruption of the normal microbiota within a body environment such asthe vagina. See Nibali et al., Journal of Oral Microbiology 6:22962,2014.

BV is diagnosed in the clinic using the Amsel Criteria and in thelaboratory using the Nugent Scoring System. The later relies on countingbacterial morphotypes with the aid of the Gram stain. In this way, theNugent Score is a visual assessment of dysbiosis—it scores the badbacteria against the good. See Nugent et al., Journal of ClinicalMicrobiology 29:297-301, 1991. The Amsel Criteria evaluates a sample forthe presence of clue cells, pH, color and odor which are key symptomsassociated with BV. See Amsel et al., Am. J. Med. 74:14-22, 1983. A wetmount of the sample is examined with a microscope to detect clue cellswhich are human epithelial cells covered with bacteria thought topredominately consist of G. vaginalis.

Molecular tests generally target multiple organisms which have strongcorrelations with bacterial vaginosis. Which organisms are targetedvaries from test to test. In nearly all cases, high abundance anaerobicbacteria are targeted such as Atopobium, Gardnerella, and Megasphaeraspecies.

The only FDA approved test for BV (BD Affirm VPIII 2010), was found tohave a sensitivity of 67.6% and a specificity of 76.4% in a study byCartwright et al. (Journal of Clinical Microbiology 51:3694-3699, 2013).For the purpose diagnosing BV, the Affirm product detects G. vaginalisas its sole indicator. The product package insert indicates the Affirmproduct is 95.1% sensitive and 83.3% specific when compared to a scoredgram stain method.

Cartwright et al., supra, used a multiplex assay for the detection ofAtopobium vaginae, BVAB-2 and Megasphaera-1 for the diagnosis of BV.They measured the performance of this assay against a combination ofNugent and Amsel results in a population of 323 women (93%African-American, 7% white non-Hispanic). They reported this test was96.9% sensitive and 92.6% specific when compared to the combination ofNugent and Amsel scores. They did not report the results of this assayrelative to the Nugent Score alone.

SUMMARY

In one aspect, the present invention provides a method for diagnosingBacterial Vaginosis (BV) in a subject. The method generally includes (a)providing a sample from a subject suspected of having BV; and (b)performing an assay for the detection of select bacterial species ineach of the genera Eggerthella and Prevotella in the sample, where theassay detects an Eggerthella species characterized by the presence of a16S rRNA gene having a nucleobase sequence that is at least 98%identical (e.g., at least 99% or 100% identical) to the sequence shownin SEQ ID NO:1 but does not detect other Eggerthella species, where theassay detects P. amnii, P. disiens, and P. bivia but does not detectother Prevotella species. The detection of at least one of Eggerthellaand Prevotella indicates BV in the subject.

In certain embodiments of a method as above, the assay for detection ofEggerthella and Prevotella is a nucleic-acid-based detection assay. Insome such embodiments, the nucleic-acid-based detection assay targetsthe 16S rRNA of Eggerthella and Prevotella. In particular variations,the nucleic-acid-based detection assay targets (i) an Eggerthella 16SrRNA region corresponding to nucleotide positions 615 to 679 of SEQ IDNO:1, and/or (ii) a Prevotella 16S rRNA region corresponding tonucleotide positions 954 to 1037 of SEQ ID NO:2. In other embodiments,the nucleic-acid-based detection assay is a non-amplification-basedassay such as, for example, a cleavage-based assay. In some suchembodiments, the cleavage-based assay detects an RNA target nucleic acidand utilizes a flap endonuclease that is capable of cleaving an RNA:DNAlinear duplex structure; in other embodiments, the cleavage-based assaydetects a DNA target nucleic acid and utilizes a flap endonuclease thatis capable of cleaving a DNA:DNA linear duplex structure. In somevariations of a method employing a nucleic-acid-based detection assay,the detection of Eggerthella and Prevotella is performed using ahomogeneous detection reaction. The detection of Eggerthella andPrevotella may further be performed in real time.

In some embodiments of a method utilizing a cleavage-based assay, theassay includes the following steps:

-   -   (i) contacting the sample with (A) an Eggerthella-specific        primer that specifically hybridizes to a target sequence within        SEQ ID NO:1, and (B) a Prevotella-specific primer that        specifically hybridizes to a target sequence within SEQ ID NO:2,        where the contacting is performed under reaction conditions        whereby each primer specifically hybridizes to its respective        16S rRNA target sequence within an Eggerthella target 16S rRNA        or a Prevotella target 16S rRNA, if present;    -   (ii) providing reactions conditions whereby the 3′ end of each        hybridized primer is extended, thereby generating a        single-stranded cDNA having a sequence complementary to a region        of the Eggerthella or Prevotella target 16S rRNA, the region        located 5′ to the respective primer target sequence;    -   (iii) contacting any Eggerthella or Prevotella cDNA from        step (ii) with (A) a first Eggerthella probe oligonucleotide        having a 3′ portion that specifically hybridizes to a first        target sequence within the Eggerthella cDNA and a 5′ portion        that does not specifically hybridize to the Eggerthella        cDNA, (B) a first Prevotella probe oligonucleotide having a 3′        portion that specifically hybridizes to a first target sequence        within the Prevotella cDNA and a 5′ portion that does not        specifically hybridize to the Prevotella cDNA, (C) a second        Eggerthella probe oligonucleotide having a 5′ portion that        specifically hybridizes to a second target sequence with the        Eggerthella cDNA, where the second Eggerthella cDNA target        sequence is located 3′ and adjacent to the first Eggerthella        cDNA target sequence, and (D) a second Prevotella probe        oligonucleotide having a 5′ portion that specifically hybridizes        to a second target sequence with the Prevotella cDNA, where the        second Prevotella cDNA target sequence is located 3′ and        adjacent to the first Prevotella cDNA target sequence,        -   where the contacting is performed under reaction conditions            whereby if the Eggerthella cDNA is present, the first and            second Eggerthella probe oligonucleotides stably hybridize            to the Eggerthella cDNA so as to form an Eggerthella linear            duplex cleavage structure, and if the Prevotella cDNA is            present, the first and second Prevotella probe            oligonucleotides stably hybridize to the Prevotella cDNA so            as to form a Prevotella linear duplex cleavage structure;    -   (iv) contacting the sample with a flap endonuclease capable of        cleaving any cleavage structure from step (iii) under reaction        conditions whereby if the Eggerthella cleavage structure is        present, cleavage of the Eggerthella cleavage structure occurs        to generate a Eggerthella cleavage product comprising the 5′        portion of the first Eggerthella probe oligonucleotide, and if        the Prevotella cleavage structure is present, cleavage of the        Prevotella cleavage structure occurs to generate a Prevotella        cleavage product comprising the 5′ portion of the first        Prevotella probe oligonucleotide; and    -   (v) detecting the presence or absence of the Eggerthella and        Prevotella cleavage products.

In some variations of a method comprising a cleavage-based assay asabove, any one or more of the following conditions is present: theEggerthella-specific primer comprises the sequence shown in SEQ ID NO:6;the Prevotella-specific primer comprises the sequence shown in SEQ IDNO:9; the 3′ portion of the first Eggerthella probe oligonucleotidecomprises the sequence shown in residues 11-27 of SEQ ID NO:4; the 3′portion of the first Prevotella probe oligonucleotide comprises thesequence shown in residues 11-25 of SEQ ID NO:7; the 5′ portion of thesecond Eggerthella probe oligonucleotide comprises the sequence shown inresidues 1-20 of SEQ ID NO:5; and/or the 5′ portion of the secondPrevotella probe oligonucleotide comprises the sequence shown inresidues 1-24 of SEQ ID NO:8.

In certain embodiments of a method comprising a cleavage-based assay asabove, detecting the Eggerthella and Prevotella cleavage productsincludes contacting the Eggerthella cleavage product with a first FRETcassette comprising a first fluorescent label and a first quencher, andcontacting the Prevotella cleavage product with a second FRET cassettecomprising a second fluorescent label and a second quencher, where eachFRET cassette hybridizes with the respective cleavage product so as toform a second Eggerthella or Prevotella cleavage structure capable ofbeing cleaved by the flap endonuclease. If the Eggerthella cleavageproduct is present, the first fluorescent label is released from thefirst FRET cassette comprising the first quencher, and if the Prevotellacleavage product is present, the second fluorescent label is releasedfrom the second FRET cassette comprising the second quencher. Thereleased first and/or second fluorescent label is then detected. In somevariations utilizing first and second FRET cassettes, the first quencherand the second quencher are the same. In some particular embodiments,the Eggerthella cleavage product includes the sequence shown in residues1-11 of SEQ ID NO:4, where residue 11 of SEQ ID NO:4 corresponds to the3′ terminal end of said cleavage product, and where the first FRETcassette optionally includes the sequence shown in SEQ ID NO:14; and/orthe Prevotella cleavage product includes the sequence shown in residues1-11 of SEQ ID NO:7, where residue 11 of SEQ ID NO:7 corresponds to the3′ terminal end of said cleavage product, and where the second FRETcassette optionally includes the sequence shown in SEQ ID NO:15.

In other embodiments of a method for diagnosing BV as above, thedetection of Eggerthella and Prevotella includes, for each target,comparing a detection signal to a predetermined detection threshold forthe target.

In some embodiments of a method for diagnosing BV in a subject, theassay for the detection of select bacterial species in each of thegenera Eggerthella and Prevotella in the sample further detects selectLactobacillus species in the subject but does not detect L. iners. Insuch embodiments, if Lactobacillus is not detected, then the detectionof at least one of Eggerthella and Prevotella indicates BV in thesubject, and if Lactobacillus is detected, then the detection of bothEggerthella and Prevotella indicates BV in the subject.

In certain embodiments of a method comprising the detection ofEggerthella, Prevotella, and Lactobacillus as above, the assay fordetection of Eggerthella, Prevotella, and Lactobacillus is anucleic-acid-based detection assay. In some such embodiments, thenucleic-acid-based detection assay targets the 16S rRNA of Eggerthella,Prevotella, and Lactobacillus. In particular variations, thenucleic-acid-based detection assay targets (i) an Eggerthella 16S rRNAregion corresponding to nucleotide positions 615 to 679 of SEQ ID NO:1,(ii) a Prevotella 16S rRNA region corresponding to nucleotide positions954 to 1037 of SEQ ID NO:2, and/or (iii) a Lactobacillus 16S rRNA regioncorresponding to nucleotide positions 837 to 944 of SEQ ID NO:3. Inother embodiments, the nucleic-acid-based detection assay is anon-amplification-based assay such as, for example, a cleavage-basedassay. In some such embodiments, the cleavage-based assay detects an RNAtarget nucleic acid and utilizes a flap endonuclease that is capable ofcleaving an RNA:DNA linear duplex structure; in other embodiments, thecleavage-based assay detects a DNA target nucleic acid and utilizes aflap endonuclease that is capable of cleaving a DNA:DNA linear duplexstructure. In some variations of a method employing a nucleic-acid-baseddetection assay, the detection of Eggerthella, Prevotella, andLactobacillus is performed using a homogeneous detection reaction. Thedetection of Eggerthella, Prevotella, and Lactobacillus may further beperformed in real time.

In some embodiments of a method utilizing a cleavage-based assay fordetection of Eggerthella, Prevotella, and Lactobacillus, the assayincludes the following steps:

-   -   (i) contacting the sample with (A) an Eggerthella-specific        primer that specifically hybridizes to a target sequence within        SEQ ID NO:1, (B) a Prevotella-specific primer that specifically        hybridizes to a target sequence within SEQ ID NO:2, and (C) a        Lactobacillus-specific primer that specifically hybridizes to a        target sequence within SEQ ID NO:3, where the contacting is        performed under reaction conditions whereby each primer        specifically hybridizes to its respective 16S rRNA target        sequence within an Eggerthella target 16S rRNA, a Prevotella        target 16S rRNA, or a Lactobacillus target 16S rRNA, if present;    -   (ii) providing reactions conditions whereby the 3′ end of each        hybridized primer is extended, thereby generating a        single-stranded cDNA having a sequence complementary to a region        of the Eggerthella, Prevotella, or Lactobacillus target 16S        rRNA, the region located 5′ to the respective primer target        sequence;    -   (iii) contacting any Eggerthella, Prevotella, or Lactobacillus        cDNA from step (ii) with (A) a first Eggerthella probe        oligonucleotide having a 3′ portion that specifically hybridizes        to a first target sequence within the Eggerthella cDNA and a 5′        portion that does not specifically hybridize to the Eggerthella        cDNA, (B) a first Prevotella probe oligonucleotide having a 3′        portion that specifically hybridizes to a first target sequence        within the Prevotella cDNA and a 5′ portion that does not        specifically hybridize to the Prevotella cDNA, (C) a        Lactobacillus probe oligonucleotide having a 3′ portion that        specifically hybridizes to a first target sequence within the        Lactobacillus cDNA and a 5′ portion that does not specifically        hybridize to the Lactobacillus cDNA, (D) a second Eggerthella        probe oligonucleotide having a 5′ portion that specifically        hybridizes to a second target sequence with the Eggerthella        cDNA, where the second Eggerthella cDNA target sequence is        located 3′ and adjacent to the first Eggerthella cDNA target        sequence, (E) a second Prevotella probe oligonucleotide having a        5′ portion that specifically hybridizes to a second target        sequence with the Prevotella cDNA, where the second Prevotella        cDNA target sequence is located 3′ and adjacent to the first        Prevotella cDNA target sequence, and (F) a second Lactobacillus        probe oligonucleotide having a 5′ portion that specifically        hybridizes to a second target sequence with the Lactobacillus        cDNA, where the second Lactobacillus cDNA target sequence is        located 3′ and adjacent to the first Lactobacillus cDNA target        sequence,        -   where the contacting is performed under reaction conditions            whereby if the Eggerthella cDNA is present, the first and            second Eggerthella probe oligonucleotides stably hybridize            to the Eggerthella cDNA so as to form an Eggerthella linear            duplex cleavage structure, if the Prevotella cDNA is            present, the first and second Prevotella probe            oligonucleotides stably hybridize to the Prevotella cDNA so            as to form a Prevotella linear duplex cleavage structure,            and if the Lactobacillus cDNA is present, the first and            second Lactobacillus probe oligonucleotides stably hybridize            to the Lactobacillus cDNA so as to form a Lactobacillus            linear duplex cleavage structure;    -   (iv) contacting the sample with a flap endonuclease capable of        cleaving any cleavage structure from step (iii) under reaction        conditions whereby if the Eggerthella cleavage structure is        present, cleavage of the Eggerthella cleavage structure occurs        to generate a Eggerthella cleavage product comprising the 5′        portion of the first Eggerthella probe oligonucleotide, if the        Prevotella cleavage structure is present, cleavage of the        Prevotella cleavage structure occurs to generate a Prevotella        cleavage product comprising the 5′ portion of the first        Prevotella probe oligonucleotide, and if the Lactobacillus        cleavage structure is present, cleavage of the Lactobacillus        cleavage structure occurs to generate a Lactobacillus cleavage        product comprising the 5′ portion of the first Lactobacillus        probe oligonucleotide; and    -   (v) detecting the presence or absence of the Eggerthella,        Prevotella, or Lactobacillus cleavage products.

In some variations of a method comprising a cleavage-based assay fordetection of Eggerthella, Prevotella, and Lactobacillus as above, anyone or more of the following conditions is present: theEggerthella-specific primer comprises the sequence shown in SEQ ID NO:6;the Prevotella-specific primer comprises the sequence shown in SEQ IDNO:9; the Lactobacillus-specific primer comprises the sequence shown inSEQ ID NO:13; the 3′ portion of the first Eggerthella probeoligonucleotide comprises the sequence shown in residues 11-27 of SEQ IDNO:4; the 3′ portion of the first Prevotella probe oligonucleotidecomprises the sequence shown in residues 11-25 of SEQ ID NO:7; the 3′portion of the first Lactobacillus probe oligonucleotide comprises thesequence shown in residues 11-27 of SEQ ID NO:10; the 5′ portion of thesecond Eggerthella probe oligonucleotide comprises the sequence shown inresidues 1-20 of SEQ ID NO:5; the 5′ portion of the second Prevotellaprobe oligonucleotide comprises the sequence shown in residues 1-24 ofSEQ ID NO:8; and/or the 5′ portion of the second Lactobacillus probeoligonucleotide comprises a sequence selected from the group consistingof (1) the sequence shown in residues 1-27 of SEQ ID NO:11 and (2) thesequence shown in residues 1-32 of SEQ ID NO:12.

In certain embodiments of a method comprising a cleavage-based assay fordetection of Eggerthella, Prevotella, and Lactobacillus as above,detecting the Eggerthella, Prevotella, and Lactobacillus cleavageproducts includes contacting the Eggerthella cleavage product with afirst FRET cassette comprising a first fluorescent label and a firstquencher, contacting the Prevotella cleavage product with a second FRETcassette comprising a second fluorescent label and a second quencher,and contacting the Lactobacillus cleavage product with a third FRETcassette comprising a third fluorescent label and a third quencher,where each FRET cassette hybridizes with the respective cleavage productso as to form a second Eggerthella, Prevotella, or Lactobacilluscleavage structure capable of being cleaved by the flap endonuclease. Ifthe Eggerthella cleavage product is present, the first fluorescent labelis released from the first FRET cassette comprising the first quencher;if the Prevotella cleavage product is present, the second fluorescentlabel is released from the second FRET cassette comprising the secondquencher; and if the Lactobacillus cleavage product is present, thethird fluorescent label is released from the third FRET cassettecomprising the third quencher. The released first, second, or thirdfluorescent label is then detected. In some variations utilizing first,second, and third FRET cassettes, the first quencher, the secondquencher, and the third quencher are the same. In some particularembodiments, the Eggerthella cleavage product includes the sequenceshown in residues 1-11 of SEQ ID NO:4, where residue 11 of SEQ ID NO:4corresponds to the 3′ terminal end of said cleavage product, and wherethe first FRET cassette optionally includes the sequence shown in SEQ IDNO:14; the Prevotella cleavage product includes the sequence shown inresidues 1-11 of SEQ ID NO:7, where residue 11 of SEQ ID NO:7corresponds to the 3′ terminal end of said cleavage product, and wherethe second FRET cassette optionally includes the sequence shown in SEQID NO:15; and/or the Lactobacillus cleavage product includes thesequence shown in residues 1-11 of SEQ ID NO:10, where residue 11 of SEQID NO:10 corresponds to the 3′ terminal end of said cleavage product,and where the third FRET cassette optionally includes the sequence shownin SEQ ID NO:16.

In certain embodiments of a method for diagnosing BV as above, themethod includes the detection of no more than ten bacterial generaassociated with BV. For example, in some embodiments, the methodincludes the detection of no more than five bacterial genera associatedwith BV, or the method does not include detection of bacterial generaassociated with BV other than Eggerthella, Prevotella, andLactobacillus.

In some embodiments of a method for diagnosing BV as above, if BV isindicated in the subject, then the method further includes administeringa treatment regime for BV to the subject. In certain embodiments, themethod is a method for monitoring BV in the subject and the subject isundergoing a treatment regime for BV prior to step (a); in some suchvariations, if BV is indicated in the subject, then the method furtherincludes either (i) administering the treatment regime for BV to thesubject (i.e., continuing to administer to same treatment regimeadministered to the subject prior to step (a)) or (ii) administering adifferent treatment regime for BV to the subject.

In other embodiments of a method for diagnosing BV and comprising thedetection of Eggerthella, Prevotella, and Lactobacillus as above, thedetection of Eggerthella, Prevotella, and Lactobacillus includes, foreach target, comparing a detection signal to a predetermined detectionthreshold for the target.

In another aspect, the present invention provides a reaction mixture fordetection of an Eggerthella target nucleic acid and a Prevotella targetnucleic acid. The reaction mixture generally includes anEggerthella-specific oligonucleotide that specifically hybridizes to atarget sequence within a target nucleic acid of an Eggerthella speciescharacterized by the presence of a 16S rRNA gene having a nucleobasesequence that is at least 98% identical (e.g., at least 99% or 100%identical) to the sequence shown in SEQ ID NO:1, but does notspecifically hybridize to a sequence within a nucleic acid from otherEggerthella species, and a Prevotella-specific oligonucleotide thatspecifically hybridizes to a target sequence within a target nucleicacid of P. amnii, P. disiens, and P. bivia, but does not specificallyhybridize to a sequence within a nucleic acid from other Prevotellaspecies.

In some embodiments of a reaction mixture as above, the Eggerthella andPrevotella target nucleic acids are 16S rRNAs of Eggerthella andPrevotella, respectively. For example, in some embodiments, (i) theEggerthella-specific oligonucleotide targets a sequence within anEggerthella 16S rRNA region corresponding to nucleotide positions 615 to679, and/or (ii) the Prevotella-specific oligonucleotide targets asequence within a Prevotella 16S rRNA region corresponding to nucleotidepositions 954 to 1037 of SEQ ID NO:2. Particularly suitableoligonucleotides targeting an Eggerthella 16S rRNA region correspondingto nucleotide positions 615 to 679 include an oligonucleotide comprisinga target-hybridizing sequence substantially corresponding to thesequence shown in SEQ ID NO:6, an oligonucleotide comprising atarget-hybridizing sequence substantially corresponding to the sequenceshown in residues 11-27 of SEQ ID NO:4, and an oligonucleotidecomprising a target-hybridizing sequence substantially corresponding tothe sequence shown in residues 1-20 of SEQ ID NO:5. Particularlysuitable oligonucleotides targeting a sequence within a Prevotella 16SrRNA region corresponding to nucleotide positions 954 to 1037 of SEQ IDNO:2 include an oligonucleotide comprising a target-hybridizing sequencesubstantially corresponding to the sequence shown in SEQ ID NO:9, anoligonucleotide comprising a target-hybridizing sequence substantiallycorresponding to the sequence shown in residues 11-25 of SEQ ID NO:7,and an oligonucleotide comprising a target-hybridizing sequencesubstantially corresponding to the sequence shown in residues 1-24 ofSEQ ID NO:8. In certain embodiments of a reaction mixture as above, themixture includes (a) at least two oligonucleotides that specificallyhybridize to two different target sequences within the Eggerthellatarget nucleic acid (e.g., at least three oligonucleotides thatspecifically hybridize to at least three different Eggerthella targetsequences), and/or (b) at least two oligonucleotides that specificallyhybridize to two different target sequences within the Prevotella targetnucleic acid (e.g., at least three oligonucleotide that specificallyhybridize to at least three different Prevotella target sequences).

In some embodiments, a reaction mixture for detection of an Eggerthellatarget nucleic acid and a Prevotella target nucleic acid includes aLactobacillus-specific oligonucleotide that specifically hybridizes to atarget sequence within a target nucleic acid of Lactobacillus species,but does not specifically hybridize to a sequence within a nucleic acidfrom L. iners. In certain variations, the Eggerthella, Prevotella, andLactobacillus target nucleic acids are 16S rRNAs of Eggerthella,Prevotella, and Lactobacillus, respectively. For example, in someembodiments, (i) the Eggerthella-specific oligonucleotide targets asequence within an Eggerthella 16S rRNA region corresponding tonucleotide positions 615 to 679, (ii) the Prevotella-specificoligonucleotide targets a sequence within a Prevotella 16S rRNA regioncorresponding to nucleotide positions 954 to 1037 of SEQ ID NO:2, and/or(iii) the Lactobacillus-specific oligonucleotide targets a sequencewithin a Lactobacillus 16S rRNA region corresponding to nucleotidepositions 837 to 944 of SEQ ID NO:3. Particularly suitableoligonucleotides targeting a sequence within an Eggerthella 16S rRNAregion corresponding to nucleotide positions 615 to 679 include anoligonucleotide comprising a target-hybridizing sequence substantiallycorresponding to the sequence shown in SEQ ID NO:6, an oligonucleotidecomprising a target-hybridizing sequence substantially corresponding tothe sequence shown in residues 11-27 of SEQ ID NO:4, and anoligonucleotide comprising a target-hybridizing sequence substantiallycorresponding to the sequence shown in residues 1-20 of SEQ ID NO:5.Particularly suitable oligonucleotides targeting a sequence within aPrevotella 16S rRNA region corresponding to nucleotide positions 954 to1037 of SEQ ID NO:2 include an oligonucleotide comprising atarget-hybridizing sequence substantially corresponding to the sequenceshown in SEQ ID NO:9, an oligonucleotide comprising a target-hybridizingsequence substantially corresponding to the sequence shown in residues11-25 of SEQ ID NO:7, and an oligonucleotide comprising atarget-hybridizing sequence substantially corresponding to the sequenceshown in residues 1-24 of SEQ ID NO:8. Particularly suitableoligonucleotides targeting a sequence within a Lactobacillus 16S rRNAregion corresponding to nucleotide positions 837 to 944 of SEQ ID NO:3include an oligonucleotide comprising a target-hybridizing sequencesubstantially corresponding to the sequence shown in SEQ ID NO:13, anoligonucleotide comprising a target-hybridizing sequence substantiallycorresponding to the sequence shown in residues 11-27 of SEQ ID NO:10,an oligonucleotide comprising a target-hybridizing sequencesubstantially corresponding to the sequence shown in residues 1-27 ofSEQ ID NO:11, and an oligonucleotide comprising a target-hybridizingsequence substantially corresponding to the sequence shown in residues1-32 of SEQ ID NO:12. In certain embodiments of a reaction mixture asabove, the mixture includes (a) at least two oligonucleotides thatspecifically hybridize to two different target sequences within theEggerthella target nucleic acid (e.g., at least three oligonucleotidesthat specifically hybridize to at least three different Eggerthellatarget sequences), (b) at least two oligonucleotide that specificallyhybridize to two different target sequences within the Prevotella targetnucleic acid (e.g., at least three oligonucleotide that specificallyhybridize to at least three different Prevotella target sequences),and/or (c) at at least two oligonucleotide that specifically hybridizeto two different target sequences within the Lactobacillus targetnucleic acid (e.g., at least three oligonucleotide that specificallyhybridize to at least three different Lactobacillus target sequences).

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise. For example, “a nucleic acid” asused herein is understood to represent one or more nucleic acids. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

“Sample” includes any specimen that may contain Eggerthella, Prevotella,or Lactobacillus or components thereof, such as nucleic acids orfragments of nucleic acids. Samples include “biological samples” whichinclude any tissue or material derived from a living or dead human thatmay contain Eggerthella, Prevotella, or Lactobacillus or componentsthereof (e.g., a target nucleic acid derived therefrom), including,e.g., vaginal swab samples, cervical brush samples, respiratory tissueor exudates such as bronchoscopy, bronchoalveolar lavage (BAL) or lungbiopsy, sputum, saliva, peripheral blood, plasma, serum, lymph node,gastrointestinal tissue, feces, urine, semen or other body fluids ormaterials. The biological sample may be treated to physically ormechanically disrupt tissue or cell structure, thus releasingintracellular components into a solution which may further containenzymes, buffers, salts, detergents and the like, which are used toprepare, using standard methods, a biological sample for analysis. Also,samples may include processed samples, such as those obtained frompassing samples over or through a filtering device, or followingcentrifugation, or by adherence to a medium, matrix, or support.

Reference to the genera “Eggerthella,” “Prevotella,” or “Lactobacillus”herein, in the particular context as targets for detection to diagnoseBV in a method of the present disclosure, and unless the context clearlydictates otherwise, is understood to mean the detection of selectspecies from these genera in accordance with the present disclosure,specifically (i) for Eggerthella, an uncultured species of Eggerthellabut not other Eggerthella species, where the uncultured Eggerthellaspecies being characterized by the presence of a 16S rRNA gene having anucleobase sequence that is at least 98% identical (e.g., at least98.5%, at least 99%, at least 99.5% or 100% identical) to the sequenceshown in SEQ ID NO:1; (ii) for Prevotella, P. amnii, P. disiens, and Pbivia, but not other Prevotella species; and (iii), for Lactobacillus,any Lactobacillus species except L. iners.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see, e.g., International PatentApplication Pub. No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties ofthe nucleic acid may be either ribose or deoxyribose, or similarcompounds having known substitutions such as, for example, 2′-methoxysubstitutions and 2′-halide substitutions (e.g., 2′-F). Nitrogenousbases may be conventional bases (A, G, C, T, U), analogs thereof (e.g.,inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham etal., 2007, BioTechniques 43: 617-24), which include derivatives ofpurine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidine bases havingsubstituent groups at the 5 or 6 position, purine bases having analtered or replacement substituent at the 2, 6 and/or 8 position, suchas 2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or3-substituted pyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825,6,949,367 and International Patent Application Pub. No. WO 93/13121,each incorporated by reference herein). Nucleic acids may include“abasic” residues in which the backbone does not include a nitrogenousbase for one or more residues (see, e.g., U.S. Pat. No. 5,585,481,incorporated by reference herein). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′-methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., Biochemistry 43:13233-41, 2004, incorporated by reference herein).Nucleic acids may include modified bases to alter the function orbehavior of the nucleic acid, e.g., addition of a 3′-terminaldideoxynucleotide to block additional nucleotides from being added tothe nucleic acid. Synthetic methods for making nucleic acids in vitroare well-known in the art although nucleic acids may be purified fromnatural sources using routine techniques.

The term “polynucleotide,” as used herein, denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “5′-to-3′,” i.e., by the addition ofnucleotides to the 3′-terminus of a growing nucleic acid.

A “nucleotide,” as used herein, is a subunit of a nucleic acidconsisting of a phosphate group, a 5-carbon sugar and a nitrogenousbase. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbonsugar is 2′-deoxyribose. The term also includes analogs of suchsubunits, such as a methoxy group at the 2′ position of the ribose(2′-O-Me).

A “non-nucleotide unit,” as used herein, is a unit that does notsignificantly participate in hybridization of a polymer. Such units mustnot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “nucleic-acid-based detection assay,” as used herein, is an assay forthe detection of a target sequence within a target nucleic acid andutilizing one more oligonucleotides that specifically hybridize to thetarget sequence.

In certain embodiments in accordance with the present invention, anucleic-acid-based detection assay is an “amplification-based assay,”i.e., an assay that utilizes one or more steps for amplifying a nucleicacid target sequence. Various amplification methods for use in detectionassays are known in the art, several of which are summarized furtherherein. For the sake of clarity, an amplification-based assay mayinclude one or more steps that do not amplify a target sequence, suchas, for example, steps used in non-amplification-based assay methods(e.g., a hybridization assay or a cleavage-based assay).

In other embodiments, a nucleic-acid-based detection assay is a“non-amplification-based assay,” i.e., an assay that does not rely onany step for amplifying a nucleic acid target sequence. For the sake ofclarity, a nucleic-acid-based detection assay that includes a reactionfor extension of a primer in the absence of any corresponding downstreamamplification oligomer (e.g., extension of a primer by a reversetranscriptase to generate an RNA:DNA duplex followed by an RNasedigestion of the RNA, resulting in a single-stranded cDNA complementaryto an RNA target but without generating copies of the cDNA) isunderstood to be a non-amplification-based assay.

An exemplary non-amplification-based assay is a “cleavage-based assay,”which is an assay that relies on the specific cleavage, by a flapendonuclease, of a linear duplex cleavage structure formed by thespecific hybridization of overlapping oligonucleotides to a targetnucleic acid. In these assays, a probe oligonucleotide containing anon-target-hybridizing flap region is cleaved in an overlap-dependentmanner by the flap endonuclease to release a cleavage product that isthen detected. The principles of cleavage-based assays are well-known inthe art, and exemplary assays are described in, for example, Lyamichevet al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn.4:135-144, 1999), Allawi et al. (J. Clin. Microbiol. 44:3443-3447,2006), U.S. Pat. Nos. 5,846,717 & 6,706,471 to Brow et al., and U.S.Pat. No. 5,614,402 to Dahlberg et al. Cleavage-based assays include,e.g., the commercially available Invader® assays (Hologic, Inc.,Madison, Wis.).

When at least a region of a first oligonucleotide and at least a regionof a second, different oligonucleotide anneal to different regions ofthe same linear complementary nucleic acid sequence, and the 3′ end ofthe annealed region of the second oligonucleotide points toward or isadjacent to the 5′ end of the annealed region of the firstoligonucleotide, the second oligonucleotide may be called the “upstream”oligonucleotide and the first oligonucleotide the “downstream”oligonucleotide.

The term “cleavage structure,” as used herein, refers to a structurethat is formed by the interaction of a probe oligonucleotide and atarget nucleic acid to form a duplex, where the resulting structure iscleavable by a flap endonuclease. The cleavage structure is a substratefor specific cleavage by the flap endonuclease, in contrast to a nucleicacid molecule that is a substrate for non-specific cleavage by agentssuch as phosphodiesterases, which cleave nucleic acid molecules withoutregard to secondary structure (i.e., no formation of a duplex structureis required).

A “flap endonuclease,” as used herein, refers to a class of nucleolyticenzymes that act as structure-specific 5′ endonucleases on nucleic acidstructures with a duplex containing a single-stranded 5′ overhang, orflap, on one of the strands that is displaced by another strand ofnucleic acid (i.e., such that there are overlapping nucleotides wherethe adjacent first and second probes hybridize to a target). A flapendonuclease may also be referred to as a “5′ endonuclease” or by theacronym “FEN” for short. FENs catalyze hydrolytic cleavage of thephosphodiester bond at the junction of single- and double-strandednucleic acid, releasing the overhang, or flap. FENs are reviewed byCeska and Savers (Trends Biochem. Sci. 23:331-336, 1998) and Liu et al.(Annu. Rev. Biochem. 73:589-615, 2004). A flap endonuclease is notrestricted to enzymes having solely 5′ nuclease activity. For example,the flap endonuclease may be a native DNA polymerase having 5′ nucleaseactivity (e.g., Taq DNA polymerase, E. coli DNA polymerase I) or amodified DNA polymerase having 5′ nuclease activity by lacking syntheticactivity (e.g., a Cleavase® enzyme).

An “overlap region” consists of the base or bases of the first probeoligonucleotide that hybridize to the target and are overlapped by thesecond probe oligonucleotide. The base on the 3′ end of the second probeoligonucleotide determines the end of the overlap region and may or maynot hybridize to the target.

A “first probe oligonucleotide,” in reference to a cleavage-baseddetection assay, refers to an oligonucleotide that interacts with atarget nucleic acid to form a cleavage structure in the presence of a“second probe oligonucleotide” that hybridizes to a region upstream ofthe first probe oligonucleotide. When annealed to the target nucleicacid, the first probe oligonucleotide and target form a cleavagestructure and cleavage by a flap endonuclease can occur within the firstprobe oligonucleotide. In the presence of an overlapping second probeoligonucleotide upstream of the first probe oligonucleotide along thetarget nucleic acid, the site of cleavage within the first probeoligonucleotide will occur after the last overlapping base (cleavagedepends on at least one overlapping base of the second probe withtarget-hybridized bases of the first probe). In addition to atarget-hybridizing region that hybridizes to a target sequence withinthe target nucleic acid, a first probe oligonucleotide contains anon-target-hybridizing region at the 5′ end (also referred to as a “flapregion”). When first and second probe oligonucleotides are annealed to atarget nucleic acid, site-specific cleavage by a flap endonucleaseoccurs to generate a cleavage product that contains the flap region andthe overlap region of the first probe oligonucleotide.

A “second probe oligonucleotide,” in reference to a cleavage-baseddetection assay, refers to an oligonucleotide that contains a sequenceat its 3′ end that, when annealed to the target nucleic acid, overlapsthe 5′ end of the target-hybridizing sequence within a downstream firstprobe oligonucleotide; typically, these regions will compete forhybridization to the same segment along a complementary target nucleicacid. The 3′ terminal nucleotide of the second probe oligonucleotide mayor may not base pair with a nucleotide in the target nucleic acid. Insome variations, only the 3′ terminal nucleotide overlaps the 5′ end ofthe target-hybridizing sequence of the first probe oligonucleotide.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleavageproduct with a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

A “target nucleic acid,” as used herein, is a nucleic acid comprising atarget sequence to be detected. Target nucleic acids may be DNA or RNAas described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence.

By “isolated” it is meant that a sample containing a target nucleic acidis taken from its natural milieu, but the term does not connote anydegree of purification.

The term “target sequence,” as used herein, refers to the particularnucleotide sequence of a target nucleic acid that is to be detected. The“target sequence” includes the complexing sequences to whicholigonucleotides (e.g., probe oligonucleotide, priming oligonucleotidesand/or promoter oligonucleotides) complex during a detection process(e.g., an amplification-based detection assay such as, for example, TMAor PCR, or a non-amplification-based detection assay such as, forexample, a 5′-endonucleose-based assay). Where the target nucleic acidis originally single-stranded, the term “target sequence” will alsorefer to the sequence complementary to the “target sequence” as presentin the target nucleic acid. Where the target nucleic acid is originallydouble-stranded, the term “target sequence” refers to both the sense (+)and antisense (−) strands. In choosing a target sequence, the skilledartisan will understand that a “unique” sequence should be chosen so asto distinguish between unrelated or closely related target nucleicacids.

“Target-hybridizing sequence” is used herein to refer to the portion ofan oligomer that is configured to hybridize with a target nucleic acidsequence. Preferably, the target-hybridizing sequences are configured tospecifically hybridize with a target nucleic acid sequence.Target-hybridizing sequences may be 100% complementary to the portion ofthe target sequence to which they are configured to hybridize; but notnecessarily. Target-hybridizing sequences may also include inserted,deleted and/or substituted nucleotide residues relative to a targetsequence. Less than 100% complementarity of a target-hybridizingsequence to a target sequence may arise, for example, when the targetnucleic acid is a plurality strains within a species, such as would bethe case for an oligomer configured to hybridize to the various strainsof Lactobacillus, but not L. iners, or configured to hybridize to P.amnii, P. disiens and P. bivia species of Prevotella. It is understoodthat other reasons exist for configuring a target-hybridizing sequenceto have less than 100% complementarity to a target nucleic acid.

Oligomer target-hybridizing sequences defined herein by reference to aspecific sequence (e.g., by reference a region within SEQ ID NO:1, 2, or3) are also understood to include functional complements thereof, unlessthe context clearly dictates otherwise. Thus, for example, where atarget-hybridizing regions of an oligomer is defined by reference to aspecific sequence corresponding to a target nucleic acid, it isunderstood that the oligomer may include a functional oligomer having atarget-hybridizing sequence that is the complement of the specificreference sequence. Or where an oligomer is defined by its configurationto hybridize to a specific sequence, it is understood that the oligomermay include a functional oligomer having a target-hybridizing sequencethat is configured to hybridize to the complement of the specificreference sequence.

The term “targets a sequence,” as used herein in reference to a regionof Eggerthella, Prevotella, or Lactobacillus nucleic acid, refers to aprocess whereby an oligonucleotide hybridizes to the target sequence ina manner that allows for detection as described herein. In oneembodiment, the oligonucleotide is complementary with the targetedEggerthella, Prevotella, or Lactobacillus nucleic acid sequence andcontains no mismatches. In another embodiment, the oligonucleotide iscomplementary but contains 1, 2, 3, 4, or 5 mismatches with the targetedEggerthella, Prevotella, or Lactobacillus nucleic acid sequence.Preferably, the oligonucleotide that hybridizes to the target nucleicacid sequence includes at least 10 to as many as 50 nucleotidescomplementary to the target sequence. It is understood that at least 10and as many as 50 is an inclusive range such that 10, 50 and each wholenumber there between are included. Preferably, the oligomer specificallyhybridizes to the target sequence.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of a referenced oligonucleotidetarget-hybridizing sequence. For example, oligonucleotides that areconfigured to specifically hybridize to a target sequence have apolynucleotide sequence that specifically hybridizes to the referencedsequence under stringent hybridization conditions.

The term “configured to specifically hybridize to” as used herein meansthat the target-hybridizing region of an oligonucleotide is designed tohave a polynucleotide sequence that could target a sequence of thereferenced Eggerthella, Prevotella, or Lactobacillus target region. Suchan oligonucleotide is not limited to targeting that sequence only, butis rather useful as a composition, in a kit or in a method for targetinga Eggerthella, Prevotella, or Lactobacillus target nucleic acid. Theoligonucleotide is designed to function as a component of an assay fordetection of Eggerthella, Prevotella, or Lactobacillus from a sample,and therefore is designed to target Eggerthella, Prevotella, orLactobacillus in the presence of other nucleic acids commonly found intesting samples. “Specifically hybridize to” does not mean exclusivelyhybridize to, as some small level of hybridization to non-target nucleicacids may occur, as is understood in the art. Rather, “specificallyhybridize to” means that the oligonucleotide is configured to functionin an assay to primarily hybridize the target so that an accuratedetection of target nucleic acid in a sample can be determined. The term“configured to” denotes an actual arrangement of the polynucleotidesequence configuration of the oligonucleotide target-hybridizingsequence.

The term “fragment,” as used herein in reference to an Eggerthella,Prevotella, or Lactobacillus targeted nucleic acid, refers to a piece ofcontiguous nucleic acid. In certain embodiments, the fragment includescontiguous nucleotides from an Eggerthella, Prevotella, or Lactobacillus16S ribosomal RNA, wherein the number of 16S contiguous nucleotides inthe fragment are less than that for the entire 16S.

The term “region,” as used herein, refers to a portion of a nucleic acidwherein said portion is smaller than the entire nucleic acid. Forexample, when the nucleic acid in reference is an oligonucleotidepromoter primer, the term “region” may be used refer to the smallerpromoter portion of the entire oligonucleotide. Similarly, and also asexample only, when the nucleic acid is a 16S ribosomal RNA, the term“region” may be used to refer to a smaller area of the nucleic acid,wherein the smaller area is targeted by one or more oligonucleotides ofthe invention. As another non-limiting example, when the nucleic acid inreference is an amplicon, the term region may be used to refer to thesmaller nucleotide sequence identified for hybridization by thetarget-hybridizing sequence of a probe.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof about 12 to 15 nt and an upper limit of about 50 to 600 nt, and otherembodiments are in a range having a lower limit of about 15 to 20 nt andan upper limit of about 22 to 100 nt. Oligonucleotides may be purifiedfrom naturally occurring sources or may be synthesized using any of avariety of well-known enzymatic or chemical methods. The termoligonucleotide does not denote any particular function to the reagent;rather, it is used generically to cover all such reagents describedherein. An oligonucleotide may serve various different functions. Forexample, it may function as a primer if it is specific for and capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase; it may function as a primerand provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Primer); and it mayfunction to detect a target nucleic acid if it is capable of hybridizingto the target nucleic acid, or an amplicon thereof, and further providesa detectible moiety (e.g., an acridinium-ester compound).

As used herein, an oligonucleotide “substantially corresponding to” aspecified reference nucleic acid sequence means that the oligonucleotideis sufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.One skilled in the art will understand that “substantially correspondingoligonucleotides” can vary from a reference sequence and still hybridizeto the same target nucleic acid sequence. It is also understood that afirst nucleic acid corresponding to a second nucleic acid includes theRNA and DNA thereof and includes the complements thereof, unless thecontext clearly dictates otherwise. This variation from the nucleic acidmay be stated in terms of a percentage of identical bases within thesequence or the percentage of perfectly complementary bases between theprobe or primer and its target sequence. Thus, in certain embodiments,an oligonucleotide “substantially corresponds” to a reference nucleicacid sequence if these percentages of base identity or complementarityare from 100% to about 80%. In preferred embodiments, the percentage isfrom 100% to about 85%. In more preferred embodiments, this percentageis from 100% to about 90%; in other preferred embodiments, thispercentage is from 100% to about 95%. Similarly, a region of a nucleicacid or amplified nucleic acid can be referred to herein ascorresponding to a reference nucleic acid sequence. One skilled in theart will understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid, and which hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a target nucleic acid and contains a 3′ OHend that is extended by a polymerase in an amplification process.Another example of an amplification oligomer is an oligomer that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. For example, the 5′ regionof an amplification oligonucleotide may include a promoter sequence thatis non-complementary to the target nucleic acid (which may be referredto as a “promoter primer” or “promoter provider”). Those skilled in theart will understand that an amplification oligomer that functions as aprimer may be modified to include a 5′ promoter sequence, and thusfunction as a promoter primer. Incorporating a 3′ blocked end furthermodifies the promoter primer, which is now capable of hybridizing to atarget nucleic acid and providing an upstream promoter sequence thatserves to initiate transcription, but does not provide a primer foroligo extension. Such a modified oligo is referred to herein as a“promoter provider” oligomer. Size ranges for amplificationoligonucleotides include those that are about 10 to about 70 nt long(not including any promoter sequence or poly-A tails) and contain atleast about 10 contiguous bases, or even at least 12 contiguous basesthat are complementary to a region of the target nucleic acid sequence(or a complementary strand thereof). The contiguous bases are at least80%, or at least 90%, or completely complementary to the target sequenceto which the amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or additionalnucleotides that participate in an amplification reaction but are notcomplementary to or contained in the target nucleic acid, or templatesequence. It is understood that when referring to ranges for the lengthof an oligonucleotide, amplicon, or other nucleic acid, that the rangeis inclusive of all whole numbers (e.g., 19-25 contiguous nucleotides inlength includes 19, 20, 21, 22, 23, 24 & 25).

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 50or more nucleotides in length. The “second region” comprises a promotersequence for an RNA polymerase. A promoter oligonucleotide is engineeredso that it is incapable of being extended by an RNA- or DNA-dependentDNA polymerase, e.g., reverse transcriptase, preferably comprising ablocking moiety at its 3′-terminus as described above. As referred toherein, a “T7 Provider” is a blocked promoter provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (see, e.g., U.S. Pat. No.4,786,600, incorporated by reference herein). PCR amplification uses aDNA polymerase, pairs of primers, and thermal cycling to synthesizemultiple copies of two complementary strands of dsDNA or from a cDNA(see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; eachincorporated by reference herein). LCR amplification uses four or moredifferent oligonucleotides to amplify a target and its complementarystrand by using multiple cycles of hybridization, ligation, anddenaturation (see, e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663, eachincorporated by reference herein). SDA uses a primer that contains arecognition site for a restriction endonuclease and an endonuclease thatnicks one strand of a hemimodified DNA duplex that includes the targetsequence, whereby amplification occurs in a series of primer extensionand strand displacement steps (see, e.g., U.S. Pat. Nos. 5,422,252;5,547,861; and 5,648,211; each incorporated by reference herein).

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refer to nucleic acid amplification that uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate. These methods generally employ an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a template complementary oligonucleotide thatincludes a promoter sequence, and optionally may include one or moreother oligonucleotides. TMA methods and single-primer transcriptionassociated amplification method are embodiments of amplification-basedassay methods used for detection of Eggerthella, Prevotella, orLactobacillus target sequences as described herein. Variations oftranscription-associated amplification are well known in the art aspreviously disclosed in detail (see, e.g., U.S. Pat. Nos. 4,868,105;5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516; and 7,374,885;and International Patent Application Pub. Nos. WO 88/01302; WO 88/10315;and WO 95/03430; each incorporated by reference herein). The person ofordinary skill in the art will appreciate that the disclosedcompositions may be used in amplification methods based on extension ofoligomer sequences by a polymerase.

The term “amplicon” or the term “amplification product,” as used herein,refers to the nucleic acid molecule generated during an amplificationprocedure that is complementary or homologous to a sequence containedwithin the target sequence. The complementary or homologous sequence ofan amplicon is sometimes referred to herein as a “target-specificsequence.” Amplicons generated using the amplification oligomers of thecurrent invention may comprise non-target specific sequences. Ampliconscan be double stranded or single stranded and can include DNA, RNA orboth. For example, DNA-dependent RNA polymerase transcribes singlestranded amplicons from double-stranded DNA duringtranscription-mediated amplification procedures. These single-strandedamplicons are RNA amplicons and can be either strand of adouble-stranded complex, depending on how the amplification oligomersare configured. Thus, amplicons can be single-stranded RNA.RNA-dependent DNA polymerases synthesize a DNA strand that iscomplementary to an RNA template. Thus, amplicons can be double-strandedDNA and RNA hybrids. RNA-dependent DNA polymerases often include RNaseactivity, or are used in conjunction with an RNase, which degrades theRNA strand. Thus, amplicons can be single stranded DNA. RNA-dependentDNA polymerases and DNA-dependent DNA polymerases synthesizecomplementary DNA strands from DNA templates. Thus, amplicons can bedouble-stranded DNA. RNA-dependent RNA polymerases synthesize RNA froman RNA template. Thus, amplicons can be double-stranded RNA.DNA-dependent RNA polymerases synthesize RNA from double-stranded DNAtemplates, also referred to as transcription. Thus, amplicons can besingle stranded RNA. Amplicons and methods for generating amplicons areknown to those skilled in the art. For convenience herein, a singlestrand of RNA or a single strand of DNA may represent an amplicongenerated by an amplification oligomer combination of the currentinvention. Such representation is not meant to limit the amplicon to therepresentation shown. Skilled artisans in possession of the instantdisclosure will use amplification oligomers and polymerase enzymes togenerate any of the numerous types of amplicons, all within the spiritand scope of the current invention.

A “non-target-specific sequence,” as used herein, refers to a region ofan oligomer sequence, wherein said region does not stably hybridize witha target sequence under standard hybridization conditions. One exampleof an oligomer with a non-target-specific sequence is a probeoligonucleotide for use in a cleavage-based assay as described herein,where the probe oligonucleotide includes a 5′ “flap” region that is notcomplementary to the target or target sequence. Other oligomers withnon-target-specific sequences include, but are not limited to, promoterprimers and molecular beacons. An amplification oligomer may contain asequence that is not complementary to the target or template sequence;for example, the 5′ region of a primer may include a promoter sequencethat is non-complementary to the target nucleic acid (referred to as a“promoter primer”). Those skilled in the art will understand that anamplification oligomer that functions as a primer may be modified toinclude a 5′ promoter sequence, and thus function as a promoter primer.Similarly, a promoter primer may be modified by removal of, or synthesiswithout, a promoter sequence and still function as a primer. A 3′blocked amplification oligomer may provide a promoter sequence and serveas a template for polymerization (referred to as a “promoter provider”).Thus, an amplicon that is generated by an amplification oligomer membersuch as a promoter primer will comprise a target-specific sequence and anon-target-specific sequence.

“Detection probe,” “detection oligonucleotide,” and “detection probeoligomer” are used interchangeably to refer to a nucleic acid oligomerthat hybridizes specifically to a target sequence in a nucleic acid, orin an amplified nucleic acid, under conditions that promotehybridization to allow detection of the target sequence or amplifiednucleic acid. Detection may either be direct (e.g., a probe hybridizeddirectly to its target sequence) or indirect (e.g., a probe linked toits target via an intermediate molecular structure). Detection probesmay be DNA, RNA, analogs thereof or combinations thereof and they may belabeled or unlabeled. Detection probes may further include alternativebackbone linkages such as, e.g., 2′-O-methyl linkages. A detectionprobe's “target sequence” generally refers to a smaller nucleic acidsequence region within a larger nucleic acid sequence that hybridizesspecifically to at least a portion of a probe oligomer by standard basepairing. A detection probe may comprise target-specific sequences andother sequences that contribute to the three-dimensional conformation ofthe probe (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412;6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. No.20060068417; each incorporated by reference herein).

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labelingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (see,e.g., U.S. Pat. Nos. 5,283,174; 5,656,207; and 5,658,737; eachincorporated by reference herein). Labels include chemiluminescentcompounds, e.g., acridinium ester (“AE”) compounds that include standardAE and derivatives (see, e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and5,639,604; each incorporated by reference herein). Synthesis and methodsof attaching labels to nucleic acids and detecting labels are wellknown. (See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Habor,N Y, 1989), Chapter 10, incorporated by reference herein. See also U.S.Pat. Nos. 5,658,737; 5,656,207; 5,547,842; 5,283,174; and 4,581,333;each incorporated by reference herein). More than one label, and morethan one type of label, may be present on a particular probe, ordetection may use a mixture of probes in which each probe is labeledwith a compound that produces a detectable signal (see, e.g., U.S. Pat.Nos. 6,180,340 and 6,350,579, each incorporated by reference herein).

“Capture probe,” “capture oligonucleotide,” and “capture probe oligomer”are used interchangeably to refer to a nucleic acid oligomer thatspecifically hybridizes to a target sequence in a target nucleic acid bystandard base pairing and joins to a binding partner on an immobilizedprobe to capture the target nucleic acid to a support. One example of acapture oligomer includes two binding regions: a sequence-binding region(e.g., target-specific portion) and an immobilized probe-binding region,usually on the same oligomer, although the two regions may be present ontwo different oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer uses a target-sequence binding regionthat includes random or non-random poly-GU, poly-GT, or poly U sequencesto bind non-specifically to a target nucleic acid and link it to animmobilized probe on a support.

As used herein, an “immobilized oligonucleotide,” “immobilized probe,”or “immobilized nucleic acid” refers to a nucleic acid binding partnerthat joins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample. Oneembodiment of an immobilized probe is an oligomer joined to a supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Supports may include known materials, such asmatrices and particles free in solution, which may be made ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal, or other compositions, of which oneembodiment is magnetically attractable particles. Supports may bemonodisperse magnetic spheres (e.g., uniform size ±5%), to which animmobilized probe is joined directly (via covalent linkage, chelation,or ionic interaction), or indirectly (via one or more linkers), wherethe linkage or interaction between the probe and support is stableduring hybridization conditions.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. Sequences that hybridize to each other may becompletely complementary or partially complementary to the intendedtarget sequence by standard nucleic acid base pairing (e.g., G:C, A:T orA:U pairing). By “sufficiently complementary” is meant a contiguoussequence that is capable of hybridizing to another sequence by hydrogenbonding between a series of complementary bases, which may becomplementary at each position in the sequence by standard base pairingor may contain one or more residues, including abasic residues that arenot complementary. Sufficiently complementary contiguous sequencestypically are at least 80%, or at least 90%, complementary to a sequenceto which an oligomer is intended to specifically hybridize. Sequencesthat are “sufficiently complementary” allow stable hybridization of anucleic acid oligomer with its target sequence under appropriatehybridization conditions, even if the sequences are not completelycomplementary. When a contiguous sequence of nucleotides of onesingle-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “completely”complementary (see, e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57, incorporated by reference herein). It is understood thatranges for percent identity are inclusive of all whole and partialnumbers (e.g., at least 90% includes 90, 91, 93.5, 97.687 and etc.).

By “preferentially hybridize” or “specifically hybridize” is meant thatunder stringent hybridization assay conditions, probes hybridize totheir target sequences, or replicates thereof, to form stableprobe:target hybrids, while at the same time formation of stableprobe:non-target hybrids is minimized Thus, a probe hybridizes to atarget sequence or replicate thereof to a sufficiently greater extentthan to a non-target sequence, to enable one having ordinary skill inthe art to accurately detect the target sequence or replicates thereof.Appropriate hybridization conditions are well-known in the art, may bepredicted based on sequence composition, or can be determined by usingroutine testing methods (see, e.g., Sambrook et al., Molecular Cloning,A Laboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51and 11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57, incorporated by reference herein).

By “nucleic acid hybrid,” “hybrid,” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

“Sample preparation” refers to any steps or method that treats a samplefor subsequent detection of Eggerthella, Prevotella, or Lactobacillus orcomponents thereof present in the sample. Sample preparation may includeany known method of concentrating components, such as microbes ornucleic acids, from a larger sample volume, such as by filtration ofairborne or waterborne particles from a larger volume sample or byisolation of microbes from a sample by using standard microbiologymethods. Sample preparation may include physical disruption and/orchemical lysis of cellular components to release intracellularcomponents into a substantially aqueous or organic phase and removal ofdebris, such as by using filtration, centrifugation or adsorption.Sample preparation may include use of a nucleic acid oligonucleotidethat selectively or non-specifically capture a target nucleic acid andseparate it from other sample components (e.g., as described in U.S.Pat. No. 6,110,678 and International Patent Application Pub. No. WO2008/016988, each incorporated by reference herein).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of a samplecomponent from other sample components.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. A primer isrequired to initiate synthesis with both RNA and DNA templates.

As used herein, a “reaction mixture” is a mixture of reagents that arecapable of reacting together to produce a product in appropriateexternal conditions over a period of time. A reaction mixture mancontain, for example, amplification assay reagents, hybridization assayreagents, and/or cleavage-based assay reagents, the recipes for whichare independently known in the art.

As used herein, a “colony-forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell. One CFUcorresponds to ˜1000 copies of rRNA.

As used herein, a “treatment regime,” in the context of a subjectdiagnosed with BV, refers to a combination of amount of a therapeuticagent administered to the subject and dosage frequency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a reference sequence for Eggerthella 16S ribosomalRNA gene (SEQ ID NO:1; uncultured bacterium clone rRNA250 16S ribosomalRNA gene, partial sequence, found at GenBank under accession numberAY959023.1 GI:66878729).

FIG. 2 illustrates a reference sequence for Prevotella 16S ribosomal RNAgene (SEQ ID NO:2; Prevotella bivia strain SEQ195 16S ribosomal RNAgene, partial sequence, found at GenBank under accession numberIN867270.1 GI:359550828).

FIG. 3 illustrates a reference sequence for Lactobacillus 16S ribosomalRNA gene (SEQ ID NO:3; Lactobacillus crispatus ST1 complete genome,strain ST1, found at GenBank under accession number FN692037.1GI:295029968).

FIG. 4 is a phylogram indicating targeted species of the genusEggerthella. Sequences obtained from uncultured species of Eggerthellawere targeted and are indicated by the box. The phylogram wasconstructed using the maximum likelihood method with a bootstrap valueof 100. The number at each branch choice indicates the frequency of thebranch choice.

FIG. 5 is a phylogram indicating targeted species of the genusPrevotella. Select sequences from the genus Prevotella were targeted andare indicated by the boxes. The phylogram was constructed using themaximum likelihood method with a bootstrap value of 100. The number ateach branch choice indicates the frequency of the branch choice.

FIG. 6 is a phylogram indicating targeted species of the genusLactobacillus. Select sequences from the genus Lactobacillus weretargeted and are indicated by the box. The phylogram was constructedusing the maximum likelihood method with a bootstrap value of 100. Thenumber at each branch point indicates the frequency of the branchchoice.

FIG. 7 is a flow diagram depicting the logic used to make bacterialvaginosis status indications from assay Velocities (V) (see Example 1).When the selected Lactobacillus Velocity is equal to or below threshold,V values for either Eggerthella or Prevotella above threshold result ina positive indication for bacterial vaginosis. When the Lactobacillus Vvalue is above the threshold, the V values for both Eggerthella andPrevotella must be above the threshold for BV to be indicated.

FIG. 8 depicts log Velocity and BV status for select species ofPrevotella showing the separation between Nugent positive and Nugentnegative samples (see Example 1). The threshold value used in theExample 1 study is indicated by the dashed red line.

FIG. 9 depicts log Velocity and BV status for select species ofEggerthella showing the separation between Nugent positive and Nugentnegative samples (see Example 1). The threshold value used in theExample 1 study is indicated by the dashed red line.

FIG. 10 depicts log Velocity and BV status for select species ofLactobacillus showing the separation between Nugent positive and Nugentnegative samples (see Example 1). The threshold value used in theExample 1 study is indicated by the dashed red line.

FIG. 11 depicts log Velocity and BV status for select species of G.vaginalis showing the separation between Nugent positive and Nugentnegative samples.

FIG. 12 depicts log Velocity and BV status for select species ofMegasphaera type 1 showing the separation between Nugent positive andNugent negative samples.

FIG. 13 depicts the relationship of log Velocity to log concentration ofa bacterial target.

DETAILED DESCRIPTION

The present invention provides methods and compositions for diagnosingBacterial Vaginosis (BV) in a subject. The methods exploithighly-specific, low abundance anaerobic bacteria belonging to thegenera Eggerthella and Prevotella. The methods generally includedetecting the presence or absence of select bacterial species in each ofthese genera in a sample from a subject suspected of having BV. Inparticular, an assay is performed for the specific detection in thesample of an uncultured species of Eggerthella but not other Eggerthellaspecies, the uncultured Eggerthella species being characterized by thepresence of a 16S rRNA gene having a nucleobase sequence that is atleast 98% identical to the sequence shown in SEQ ID NO:1, and an assayfor the specific detection in the sample of P. amnii, P. disiens, and Pbivia, but not other Prevotella species. Utilizing thesespecies-specific assays, the detection of at least one of Eggerthellaand Prevotella in the sample is generally indicative of BV in thesubject, with greater sensitivity and specificity than some existingtests.

The performance of the Eggerthella/Prevotella combination for diagnosingBV can be improved by the inclusion of Lactobacillus as an indicator ofvaginal health. Accordingly, in some embodiments, the method furtherincludes detecting the presence or absence of select species ofLactobacillus. In particular, an assay is performed for the specificdetection in the sample of Lactobacillus species, where the assay doesnot detect L. iners. In these embodiments, if Lactobacillus is notdetected, then the detection of either Eggerthella or Prevotellaindicates BV in the subject, and if Lactobacillus is detected, then thedetection of both Eggerthella and Prevotella indicates BV in thesubject. As described further herein, an exemplary assay using thiscombination of bacterial targets and logic yielded a test that was 95.6%sensitive and 97.3% specific when compared to the Nugent Score.

In some embodiments, the uncultured Eggerthella species is characterizedby the presence of a 16S rRNA gene having a nucleobase sequence that isat least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, at least 99.9%, or 100% identical to the sequenceshown in SEQ ID NO:1. Typically, the 16S rRNA gene of the unculturedEggerthella species has a region that is 100% identical to nucleotidepositions 615 to 679 of SEQ ID NO:1.

While the select bacterial species from Eggerthella, Prevotella, and/orLactobacillus may be detected using any suitable method, it is presentlypreferred that the select species are detected using anucleic-acid-based detection assay. Nucleic-acid-based detection assaysin accordance with the present invention generally utilizeoligonucleotides that specifically hybridize to a target nucleic acid ofthe select species of Eggerthella, Prevotella, or Lactobacillus withminimal cross-reactivity to other nucleic acids suspected of being in asample. As previously indicated, an assay to detect the unculturedEggerthella species does not detect other Eggerthella species; an assayto detect P. amnii, P. disiens, and P bivia does not detect otherPrevotella species; and an assay to detect Lactobacillus species doesnot detect L. iners. Accordingly, oligonucleotides fornucleic-acid-based detection of the select species of Eggerthella,Prevotella, or Lactobacillus will specifically hybridize to the targetspecies within the respective genus with minimal cross-reactivity tonon-target species. Additionally, oligonucleotides fornucleic-acid-based detection of the select species of Eggerthella,Prevotella, and Lactobacillus will have minimal cross-reactivity tospecies within other bacterial genera, including, for example,Trichomonas sp.; Trichomonas vaginalis; Candida sp.; Bacterium from theorder Clostridiales; Clostridium-like sp.; Atopobium sp.; Atopobiumvaginae; Enterobacteria; Peptostreptococcus micros; Aerococcuschristensenii; Leptotrichia amnionii; Peptoniphilus sp.; Dialister sp.;Mycoplasma hominis; Sneathia sanguinegens; Anaerococcus tetradius;Mobiluncus sp.; Mobiluncus hominis; Megasphaera sp.; Leptotrichiasanguinegens; and Finegoldia magna. In one aspect, a nucleic-acid-baseddetection assay in accordance with the present invention furtherincludes components for detecting one of more of these organisms, orother bacterial genera associated with BV.

In particular embodiments, a nucleic-acid-based detection assay targetsthe 16S rRNA of Eggerthella, Prevotella, and/or Lactobacillus, or a geneencoding the 16S rRNA. Particularly suitable target regions of the 16SrRNA or the encoding gene are (i) an Eggerthella 16S rRNA regioncorresponding to nucleotide positions 615 to 679 of SEQ ID NO:1; (ii) aPrevotella 16S rRNA region corresponding to nucleotide positions 954 to1037 of SEQ ID NO:2; and (iii) a Lactobacillus 16S rRNA regioncorresponding to nucleotide positions 837 to 944 of SEQ ID NO:3. Inspecific variations of a nucleic-acid-based detection assay targeting a16S rRNA region as above, (a) an Eggerthella-specific oligonucleotideincludes a target-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:6, a sequencesubstantially corresponding to the sequence shown in residues 11-27 ofSEQ ID NO:4, or a sequence substantially corresponding to the sequenceshown in residues 1-20 of SEQ ID NO:5; (b) a Prevotella-specificoligonucleotide includes a target-hybridizing region comprising asequence substantially corresponding to the sequence shown in SEQ IDNO:9, a sequence substantially corresponding to the sequence shown inresidues 11-25 of SEQ ID NO:7, or a sequence substantially correspondingto the sequence shown in residues 1-24 of SEQ ID NO:8; and/or (c) aLactobacillus-specific oligonucleotide includes a target-hybridizingregion comprising a sequence substantially corresponding to the sequenceshown in SEQ ID NO:13, a sequence substantially corresponding to thesequence shown in residues 11-27 of SEQ ID NO:10, a sequencesubstantially corresponding to the sequence shown in residues 1-27 ofSEQ ID NO:11, or a sequence substantially corresponding to the sequenceshown in residues 1-32s of SEQ ID NO:12. In some such embodiments, (a)an Eggerthella-specific oligonucleotide includes a target-hybridizingregion comprising or consisting of the sequence shown in SEQ ID NO:6,the sequence shown in residues 11-27 of SEQ ID NO:4, or the sequenceshown in residues 1-20 of SEQ ID NO:5; (b) a Prevotella-specificoligonucleotide includes a target-hybridizing region comprising orconsisting of the sequence shown in SEQ ID NO:9, the sequence shown inresidues 11-25 of SEQ ID NO:7, or the sequence shown in residues 1-24 ofSEQ ID NO:8; and/or (c) a Lactobacillus-specific oligonucleotideincludes a target-hybridizing region comprising or consisting of thesequence shown in SEQ ID NO:13, the sequence shown in residues 11-27 ofSEQ ID NO:10, the sequence shown in residues 1-27 of SEQ ID NO:11, orthe sequence shown in residues 1-32s of SEQ ID NO:12. In certainembodiments, a nucleic-acid-based detection assay utilizes at least twoor three Eggerthella-specific oligonucleotides, at least two or threePrevotella-specific oligonucleotides, and/or at least two or threeLactobacillus-specific oligonucleotides, which may be oligonucleotidesselected from those specified above.

In some embodiments of a method comprising the use of anucleic-acid-base detection assay, an amplification-based assay is usedto detect the select bacterial species of Eggerthella, Prevotella,and/or Lactobacillus. Such variations generally include amplifying atarget sequence within a bacterial target nucleic acid utilizing an invitro nucleic acid amplification reaction and detecting the amplifiedproduct by, for example, specifically hybridizing the amplified productwith a nucleic acid detection probe that provides a signal to indicatethe presence of a select bacterial species in the sample. Theamplification step includes contacting the sample with two or moreamplification oligomers specific for a target sequence in a targetnucleic acid (e.g., a target sequence in a 16S rRNA) to produce anamplified product if the target nucleic acid is present in the sample.Amplification synthesizes additional copies of the target sequence orits complement by using at least one nucleic acid polymerase to extendthe sequence from an amplification oligomer (a primer) using a templatestrand. One embodiment for detecting the amplified product uses ahybridizing step that includes contacting the amplified product with atleast one probe specific for a sequence amplified by the selectedamplification oligomers, e.g., a sequence contained in the targetsequence flanked by a pair of selected primers. Suitable amplificationmethods include, for example, replicase-mediated amplification,polymerase chain reaction (PCR), ligase chain reaction (LCR),strand-displacement amplification (SDA), and transcription-mediated ortranscription-associated amplification (TMA). Such amplification methodsare well-known in the art (see, e.g., discussion of amplificationmethods in Definitions section, supra) and are readily used inaccordance with the methods of the present invention.

For example, some amplification methods that use TMA amplificationinclude the following steps. Briefly, the target nucleic acid thatcontains the sequence to be amplified is provided as single strandednucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art willappreciate that conventional melting of double stranded nucleic acid(e.g., dsDNA) may be used to provide single-stranded target nucleicacids. A promoter primer binds specifically to the target nucleic acidat its target sequence and a reverse transcriptase (RT) extends the 3′end of the promoter primer using the target strand as a template tocreate a cDNA copy of the target sequence strand, resulting in anRNA:DNA duplex. An RNase digests the RNA strand of the RNA:DNA duplexand a second primer binds specifically to its target sequence, which islocated on the cDNA strand downstream from the promoter primer end. RTsynthesizes a new DNA strand by extending the 3′ end of the secondprimer using the first cDNA template to create a dsDNA that contains afunctional promoter sequence. An RNA polymerase specific for thepromoter sequence then initiates transcription to produce RNAtranscripts that are about 100 to 1000 amplified copies (“amplicons”) ofthe initial target strand in the reaction. Amplification continues whenthe second primer binds specifically to its target sequence in each ofthe amplicons and RT creates a DNA copy from the amplicon RNA templateto produce an RNA:DNA duplex. RNase in the reaction mixture digests theamplicon RNA from the RNA:DNA duplex and the promoter primer bindsspecifically to its complementary sequence in the newly synthesized DNA.RT extends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds totranscribe additional amplicons that are complementary to the targetstrand. The autocatalytic cycles of making more amplicon copies repeatduring the course of the reaction resulting in about a billion-foldamplification of the target nucleic acid present in the sample. Theamplified products may be detected in real-time during amplification, orat the end of the amplification reaction by using a probe that bindsspecifically to a target sequence contained in the amplified products.Detection of a signal resulting from the bound probes indicates thepresence of the target nucleic acid in the sample.

In some embodiments, the method utilizes a “reverse” TMA reaction. Insuch variations, the initial or “forward” amplification oligomer is apriming oligonucleotide that hybridizes to the target nucleic acid inthe vicinity of the 3′-end of the target region. A reverse transcriptase(RT) synthesizes a cDNA strand by extending the 3′-end of the primerusing the target nucleic acid as a template. The second or “reverse”amplification oligomer is a promoter primer or promoter provider havinga target-hybridizing sequence configure to hybridize to atarget-sequence contained within the synthesized cDNA strand. Where thesecond amplification oligomer is a promoter primer, RT extends the 3′end of the promoter primer using the cDNA strand as a template to createa second, cDNA copy of the target sequence strand, thereby creating adsDNA that contains a functional promoter sequence. Amplification thencontinues essentially as described above for initiation of transcriptionfrom the promoter sequence utilizing an RNA polymerase. Alternatively,where the second amplification oligomer is a promoter provider, aterminating oligonucleotide, which hybridizes to a target sequence thatis in the vicinity to the 5′-end of the target region, is typicallyutilized to terminate extension of the priming oligomer at the 3′-end ofthe terminating oligonucleotide, thereby providing a defined 3′-end forthe initial cDNA strand synthesized by extension from the primingoligomer. The target-hybridizing sequence of the promoter provider thenhybridizes to the defined 3′-end of the initial cDNA strand, and the3′-end of the cDNA strand is extended to add sequence complementary tothe promoter sequence of the promoter provider, resulting in theformation of a double-stranded promoter sequence. The initial cDNAstrand is then used a template to transcribe multiple RNA transcriptscomplementary to the initial cDNA strand, not including the promoterportion, using an RNA polymerase that recognizes the double-strandedpromoter and initiates transcription therefrom. Each of these RNAtranscripts is then available to serve as a template for furtheramplification from the first priming amplification oligomer.

In certain embodiments comprising an amplification-based detectionassay, a combination of at least two amplification oligomers is utilizedfor the detection of an Eggerthella 16S rRNA or a gene encoding anEggerthella 16S rRNA. The oligomer combination may include first andsecond amplification oligomers for amplifying an Eggerthella nucleicacid target region corresponding to SEQ ID NO:1 from about nucleotideposition 615 to about nucleotide position 679. For example, in someembodiments, the first amplification oligomer includes atarget-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:6, and the secondamplification oligomer includes a target-hybridizing region comprising asequence substantially corresponding to the sequence shown in residues11-27 of SEQ ID NO:4, or a sequence substantially corresponding to thesequence shown in residues 1-20 of SEQ ID NO:5. In more particularvariations, the first amplification oligomer includes atarget-hybridizing region comprising or consisting of the sequence shownin SEQ ID NO:6, and the second amplification oligomer includes atarget-hybridizing region comprising or consisting of the sequence shownin residues 11-27 of SEQ ID NO:4, or comprising or consisting of thesequence shown in residues 1-20 of SEQ ID NO:5.

In certain embodiments comprising an amplification-based detectionassay, a combination of at least two amplification oligomers is utilizedfor the detection of a Prevotella 16S rRNA or a gene encoding aPrevotella 16S rRNA. The oligomer combination may include first andsecond amplification oligomers for amplifying a Prevotella nucleic acidtarget region corresponding to SEQ ID NO:2 from about nucleotideposition 954 to about nucleotide position 1034. For example, in someembodiments, the first amplification oligomer includes atarget-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:9, and the secondamplification oligomer includes a target-hybridizing region comprising asequence substantially corresponding to the sequence shown in residues11-25 of SEQ ID NO:7, or a sequence substantially corresponding to thesequence shown in residues 1-24 of SEQ ID NO:8. In more particularvariations, the first amplification oligomer includes atarget-hybridizing region comprising or consisting of the sequence shownin SEQ ID NO:9, and the second amplification oligomer includes atarget-hybridizing region comprising or consisting of the sequence shownin residues 11-25 of SEQ ID NO:7, or comprising or consisting of thesequence shown in residues 1-24 of SEQ ID NO:8.

In certain aspects comprising an amplification-based detection assay, acombination of at least two amplification oligomers is utilized for thedetection of a Lactobacillus 16S rRNA or a gene encoding a Lactobacillus16S rRNA. The oligomer combination may include first and secondamplification oligomers for amplifying a Lactobacillus nucleic acidtarget region corresponding to SEQ ID NO:3 from about nucleotideposition 837 to about nucleotide position 944. For example, in someembodiments, the first amplification oligomer includes atarget-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:13, and the secondamplification oligomer includes a target-hybridizing region comprising asequence substantially corresponding to the sequence shown in residues11-27 of SEQ ID NO:10, a sequence substantially corresponding to thesequence shown in residues 1-27 of SEQ ID NO:11, or a sequencesubstantially corresponding to the sequence shown in residues 1-32 ofSEQ ID NO:12. In more particular variations, the first amplificationoligomer includes a target-hybridizing region comprising or consistingof the sequence shown in SEQ ID NO:13, and the second amplificationoligomer includes a target-hybridizing region comprising or consistingof the sequence shown in residues 11-27 of SEQ ID NO:10, comprising orconsisting of the sequence shown in residues 1-27 of SEQ ID NO:11, orcomprising or consisting of the sequence shown in residues 1-32 of SEQID NO:12.

Detection of the amplified products may be accomplished by a variety ofmethods to detect a signal specifically associated with the amplifiedtarget sequence. The nucleic acids may be associated with a surface thatresults in a physical change, such as a detectable electrical change.Amplified nucleic acids may be detected by concentrating them in or on amatrix and detecting the nucleic acids or dyes associated with them(e.g., an intercalating agent such as ethidium bromide or cyber green),or detecting an increase in dye associated with nucleic acid in solutionphase. Other methods of detection may use nucleic acid detection probesthat are configured to specifically hybridize to a sequence in theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481; each incorporated by referenceherein). Directly or indirectly labeled probes that specificallyassociate with the amplified product provide a detectable signal thatindicates the presence of the target nucleic acid in the sample. Forexample, if the target nucleic acid is the 16S rRNA of Eggerthella,Prevotella, or Lactobacillus, the amplified product will contain atarget sequence in or complementary to a sequence in the 16S rRNA, and aprobe will bind directly or indirectly to a sequence contained in theamplified product to indicate the presence of the 16S rRNA ofEggerthella, Prevotella, or Lactobacillus in the tested sample.

Detection probes that hybridize to the complementary amplified sequencesmay be DNA or RNA oligomers, or oligomers that contain a combination ofDNA and RNA nucleotides, or oligomers synthesized with a modifiedbackbone, e.g., an oligomer that includes one or more 2′-methoxysubstituted ribonucleotides. Probes used for detection of the amplifiedsequences may be unlabeled and detected indirectly (e.g., by binding ofanother binding partner to a moiety on the probe) or may be labeled witha variety of detectable labels. In some embodiments of the method fordiagnosing BV, such as in certain embodiments usingtranscription-mediated amplification (TMA), the detection probe is alinear chemiluminescently labeled probe such as, e.g., a linearacridinium ester (AE) labeled probe.

The detection step may also provide additional information on theamplified sequence, such as, e.g., all or a portion of its nucleic acidbase sequence. Detection may be performed after the amplificationreaction is completed, or may be performed simultaneously withamplifying the target region, e.g., in real time. In one embodiment, thedetection step allows homogeneous detection, e.g., detection of thehybridized probe without removal of unhybridized probe from the mixture(see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174, each incorporated byreference herein).

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. One example of such detection uses a luminescentally labeledprobe that hybridizes to target nucleic acid. Luminescent label is thenhydrolyzed from non-hybridized probe. Detection is performed bychemiluminescence using a luminometer. (see, e.g., International PatentApplication Pub. No. WO 89/002476, incorporated by reference herein). Inother embodiments that use real-time detection, the detection probe maybe a hairpin probe such as, for example, a molecular beacon, moleculartorch, or hybridization switch probe that is labeled with a reportermoiety that is detected when the probe binds to amplified product. Suchprobes may comprise target-hybridizing sequences andnon-target-hybridizing sequences. Various forms of such probes have beendescribed previously (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728;5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945;and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1;each incorporated by reference herein).

In some embodiments of a method comprising the use of anucleic-acid-base detection assay, a non-amplification-based assay isused to detect the select bacterial species of Eggerthella, Prevotella,and/or Lactobacillus. In some such embodiments, thenon-amplification-based assay is a hybridization assay comprising thehybridization of a specific detection probe to a target nucleic acid.Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known, including those referred to in,e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual (3rd ed.Cold Spring Harbor, N.Y., 2002), and Berger and Kimmel, Methods inEnzymology, Vol. 152, Guide to Molecular Cloning Techniques (AcademicPress, Inc., San Diego, Calif., 1987). Generally, the probe and sampleare mixed under conditions that will permit specific nucleic acidhybridization, and specific hybridization of the probe to its respectivetarget is then detected. Nucleic acid hybridization is adaptable to avariety of assay formats. One suitable format is the sandwich assayformat, which is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support, which has adsorbed to it or covalently coupled to itimmobilized nucleic acid probe that is unlabeled and complementary toone portion of the DNA sequence. Target nucleic acid is hybridized tothe immobilized probe, and a second, labeled detection probe—which iscomplementary to a second and different region of the same DNA strand towhich the immobilized, unlabeled nucleic acid probe is hybridized—ishybridized to the [target nucleic acid]:[immobilized probe] duplex todetect the target nucleic acid. Another exemplary format utilizeselectrochemical detection of target nucleic acids hybridized tounlabeled detection probes immobilized on a suitable electrode surfaceas a signal transducer. See, e.g., Drummond et al., Nat. Biotechnol.21:1192, 2003; Gooding, Electroanalysis 14:1149, 2002; Wang, Anal. Chim.Acta 469:63, 2002; Cagnin et al., Sensors 9:3122, 2009; Katz andWillner, Electroanalysis 15:913, 2003; Daniels and Pourmand,Electroanalysis 19:1239, 2007.

In certain embodiments comprising a hybridization assay, a detectionprobe is utilized for the detection of an Eggerthella, Prevotella,and/or Lactobacillus 16S rRNA or a gene encoding an Eggerthella,Prevotella, and/or Lactobacillus 16S rRNA. In such embodiments, a probefor detecting an Eggerthella 16S rRNA or gene encoding an Eggerthella16S rRNA specifically hybridizes to a nucleic acid target regioncorresponding to SEQ ID NO:1 from about nucleotide position 615 to aboutnucleotide position 679; a probe for detecting a Prevotella 16S rRNA orgene encoding a Prevotella 16S rRNA specifically hybridizes to a nucleicacid target region corresponding to SEQ ID NO:2 from about nucleotideposition 954 to about nucleotide position 1034; and/or a probe fordetecting a Lactobacillus 16S rRNA or gene encoding a Lactobacillus 16SrRNA specifically hybridizes to a nucleic acid target regioncorresponding to SEQ ID NO:3 from about nucleotide position 837 to aboutnucleotide position 944. For example, in some variations, a probe fordetection of Eggerthella includes a target-hybridizing region comprisinga sequence substantially corresponding to the sequence shown in SEQ IDNO:6, a sequence substantially corresponding to the sequence shown inresidues 11-27 of SEQ ID NO:4, or a sequence substantially correspondingto the sequence shown in residues 1-20 of SEQ ID NO:5 (e.g., atarget-hybridizing region comprising or consisting of the sequence shownin SEQ ID NO:6, residues 11-27 of SEQ ID NO:4, or residues 1-20 of SEQID NO:5). In some variations, a probe for detection of Prevotellaincludes a target-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:9, a sequencesubstantially corresponding to the sequence shown in residues 11-25 ofSEQ ID NO:7, or a sequence substantially corresponding to the sequenceshown in residues 1-24 of SEQ ID NO:8 (e.g., a target-hybridizing regioncomprising or consisting of the sequence shown in SEQ ID NO:9, residues11-25 of SEQ ID NO:7, or residues 1-24 of SEQ ID NO:8). In somevariations, a probe for detection of Lactobacillus includes atarget-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:13, a sequencesubstantially corresponding to the sequence shown in residues 11-27 ofSEQ ID NO:10, a sequence substantially corresponding to the sequenceshown in residues 1-27 of SEQ ID NO:11, or a sequence substantiallycorresponding to the sequence shown in residues 1-32 of SEQ ID NO:12(e.g., a target-hybridizing region comprising or consisting of thesequence shown in SEQ ID NO:13, residues 11-27 of SEQ ID NO:10, residues1-27 of SEQ ID NO:11, or residues 1-32 of SEQ ID NO:12).

In some preferred embodiments, a non-amplification-based assay fordetection of Eggerthella, Prevotella, and/or Lactobacillus is acleavage-based assay, in which a probe oligonucleotide containing anon-target-hybridizing flap region is cleaved in an overlap-dependentmanner by a flap endonuclease to release a cleavage product that is thendetected. Exemplary cleavage-based assay reagents are described in,e.g., Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al.(Mol. Diagn. 4:135-144, 1999), and Allawi et al. (J. Clin. Microbiol.44:3443-3447, 2006). Appropriate conditions for flap endonucleasereactions are either known or can be readily determined using methodsknown in the art (see, e.g., Kaiser et al., J. Biol. Chem.274:2138-721394, 1999). Exemplary flap endonucleases that may be used inthe method include Thermus aquaticus DNA polymerase I, Thermusthermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidusFEN-1, Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1,Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1,CLEAVASE® (Hologic, Inc., Madison, Wis.), S. cerevisiae RTH1, S.cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′exonuclease, Pyrococcus horikoshii FEN-1, human endonuclease 1, calfthymus 5′-3′ exonuclease, including homologs thereof in eubacteria,eukaryotes, and archaea, such as members of the class II family ofstructure-specific enzymes, as well as enzymatically active mutants orvariants thereof. Descriptions of flap endonucleases can be found in,for example, Lyamichev et al., Science 260:778-783, 1993; Eis et al.,Nat. Biotechnol. 19:673-676, 2001; Shen et al., Trends in Bio. Sci.23:171-173, 1998; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999;Ma et al., J. Biol. Chem. 275:24693-24700, 2000; Allawi et al., J. Mol.Biol. 328:537-554, 2003; Sharma et al., J. Biol. Chem. 278:23487-23496,2003; and Feng et al., Nat. Struct. Mol. Biol. 11:450-456, 2004.

In certain variations, a cleavage-based assay detects an RNA targetnucleic acid of Eggerthella, Prevotella, and/or Lactobacillus, and thecleavage-based assay utilizes a flap endonuclease that is capable ofcleaving and RNA:DNA linear duplex structure. In some alternativeembodiments, a cleavage-based assay detects a DNA target nucleic acid ofEggerthella, Prevotella, and/or Lactobacillus, and the cleavage-basedassay utilizes a flap endonuclease that is capable of cleaving andDNA:DNA linear duplex structure. Exemplary flap endonucleases capable ofcleaving RNA:DNA duplexes include polymerase-deficient 5′ nucleases ofthe genus Thermus as well as certain CLEAVASE® enzymes (Hologic, Inc.,Madison, Wis.) such as, for example, CLEAVASE® BN (BstX-NotI deletion ofTaq polymerase, see U.S. Pat. No. 5,614,402), CLEAVASE® II (“AG” mutantof full length Taq polymerase, see U.S. Pat. No. 5,614,402), CLEAVASE®VII (synthesis-deficient mutation of full length Thermus thermophiluspolymerase), CLEAVASE® IX (polymerase deficient mutant of the Tth DNApolymerase), and CLEAVASE® XII (polymerase deficient chimeric polymeraseconstructed from fragments of taq DNA polymerase and Tth DNApolymerase). Exemplary flap endonucleases capable of cleaving DNA:DNAduplexes include the flap endonucleases indicated above, as well asCLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1(Archaeoglobus fulgidus FEN-1 with 6 histidines on the C-terminus),CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1(Archaeoglobus veneficus FEN-1 with 6 histidines on the C-terminus).

In some embodiments, a cleavage-based assay detects an RNA targetnucleic acid of Eggerthella, Prevotella, and/or Lactobacillus, and theassay includes a step for synthesizing a DNA complement of an RNA targetregion, which cDNA strand is then hybridized to overlapping first andsecond probe oligonucleotides to form a linear duplex cleavage structurefor cleavage by the flap endonuclease. Reaction conditions forsynthesizing cDNA from an RNA template, using an RNA-dependent DNApolymerase (reverse transcriptase), are well-known in the art.

In some embodiments, a cleavage-based assay targets an Eggerthella,Prevotella,and/or Lactobacillus 16S rRNA or a gene encoding anEggerthella, Prevotella, and/or Lactobacillus 16S rRNA. In certainvariations, a cleavage-based assay targets (i) an Eggerthella 16S rRNAregion corresponding to nucleotide positions 615 to 679 of SEQ ID NO:1,(ii) a Prevotella 16S rRNA region corresponding to nucleotide positions954 to 1037 of SEQ ID NO:2, and/or (iii) a Lactobacillus 16S rRNA regioncorresponding to nucleotide positions 837 to 944 of SEQ ID NO:3.

For example, in certain embodiments of a cleavage-based assay targetingan Eggerthella 16S rRNA target region, utilizing overlapping first andsecond oligonucleotides, the first probe oligonucleotide includes atarget-hybridizing region substantially corresponding to the sequenceshown in residues 11-27 of SEQ ID NO:4 and/or the second probeoligonucleotide includes a target-hybridizing region substantiallycorresponding to the sequence shown in residues 1-20 of SEQ ID NO:5. Insome variations, a reverse transcriptase reaction is performed tosynthesize a cDNA copy of the 16S rRNA, such as, for example, a reversetranscriptase reaction utilizing a primer having a target-hybridizingregion substantially corresponding to the sequence shown in SEQ ID NO:6.In more particular variations for the detection of Eggerthella, a firstprobe oligonucleotide includes a target-hybridizing region comprising orconsisting of the sequence shown in residues 11-27 of SEQ ID NO:4; asecond probe oligonucleotide includes a target-hybridizing regioncomprising or consisting of the sequence shown in residues 1-20 of SEQID NO:5; and/or a reverse transcriptase primer includes atarget-hybridizing sequence comprising or consisting of the sequenceshown in SEQ ID NO:6.

In some embodiments of a cleavage-based assay targeting an Prevotella16S rRNA target region, utilizing overlapping first and secondoligonucleotides, the first probe oligonucleotide includes atarget-hybridizing region substantially corresponding to the sequenceshown in residues 11-25 of SEQ ID NO:7 and/or the second probeoligonucleotide includes a target-hybridizing region substantiallycorresponding to the sequence shown in residues 1-24 of SEQ ID NO:8. Insome variations, a reverse transcriptase reaction is performed tosynthesize a cDNA copy of the 16S rRNA, such as, for example, a reversetranscriptase reaction utilizing a primer having a target-hybridizingregion substantially corresponding to the sequence shown in SEQ ID NO:9.In more particular variations for the detection of Prevotella, a firstprobe oligonucleotide includes a target-hybridizing region comprising orconsisting of the sequence shown in residues 11-25 of SEQ ID NO:7; asecond probe oligonucleotide includes a target-hybridizing regioncomprising or consisting of the sequence shown in residues 1-24 of SEQID NO: 8; and/or a reverse transcriptase primer includes atarget-hybridizing sequence comprising or consisting of the sequenceshown in SEQ ID NO:9.

In some embodiments of a cleavage-based assay targeting an Lactobacillus16S rRNA target region, utilizing overlapping first and secondoligonucleotides, the first probe oligonucleotide includes atarget-hybridizing region substantially corresponding to the sequenceshown in residues 11-27 of SEQ ID NO:10 and/or the second probeoligonucleotide includes a target-hybridizing region substantiallycorresponding to a sequence selected from the sequence shown in residues1-27 of SEQ ID NO:11 and the sequence shown is residues 1-32 of SEQ IDNO:12. In some variations, a reverse transcriptase reaction is performedto synthesize a cDNA copy of the 16S rRNA, such as, for example, areverse transcriptase reaction utilizing a primer having atarget-hybridizing region substantially corresponding to the sequenceshown in SEQ ID NO:13. In more particular variations for the detectionof Lactobacillus, a first probe oligonucleotide includes atarget-hybridizing region comprising or consisting of the sequence shownin residues 11-27 of SEQ ID NO:10; a second probe oligonucleotideincludes a target-hybridizing region comprising or consisting of asequence selected from the sequence shown in residues 1-27 of SEQ IDNO:11 and the sequence shown is residues 1-32 of SEQ ID NO:12; and/or areverse transcriptase primer includes a target-hybridizing sequencecomprising or consisting of the sequence shown in SEQ ID NO:13.

In typical variations of a cleavage-based detection assay, a cleavageproduct is detected using a hairpin oligonucleotide probe known as aFRET cassette, which contains a fluorophore at its 5′ end and a nearbyquencher that quenches the fluorophore. Hybridization of the cleavageproduct with a FRET cassette produces a secondary substrate for the flapendonuclease, whereby the 5′ fluorophore-containing base is cleaved fromthe cassette, thereby generating a fluorescence signal. Principlesgoverning the design and construction of FRET cassettes for use incleavage-based assays are well-known in the art, and these principlesmay be readily adapted by a skilled artisan for using such probes inaccordance with certain embodiments of the present invention. Inspecific embodiments, (i) where an Eggerthella cleavage productcomprises the sequence shown in residues 1-11 of SEQ ID NO:4 and residue11 corresponds to the 3′ terminal end of the cleavage product, a FRETcassette for detection of the Eggerthella cleavage product comprises orconsists of the sequence shown in SEQ ID NO:14; (ii) where a Prevotellacleavage product comprises the sequence shown in residues 1-11 of SEQ IDNO:7 and residue 11 corresponds to the 3′ terminal end of the cleavageproduct, a FRET cassette for detection of the Prevotella cleavageproduct comprises or consists of the sequence shown in SEQ ID NO:15;and/or (iii) where a Lactobacillus cleavage product comprises thesequence shown in residues 1-11 of SEQ ID NO:10 and residue 11corresponds to the 3′ terminal end of the cleavage product, a FRETcassette for detection of the Lactobacillus cleavage product comprisesor consists of the sequence shown in SEQ ID NO:16. The secondarysubstrate formed by hybridization of a FRET cassette to an Eggerthellacleavage product, a Prevotella cleavage product, or a Lactobacilluscleavage product (each comprising a 5′ portion of a first Eggerthellaprobe oligonucleotide, a first Prevotella probe oligonucleotide, or afirst Lactobacillus probe oligonucleotide, respectively) is alsoreferred to herein as a “second Eggerthella cleavage structure,” a“second Prevotella cleavage structure,” or a “second Lactobacilluscleavage structure,” respectively. For the sake of clarity, the use ofthe term “second [Eggerthella, Prevotella, or Lactobacillus] cleavagestructure” in this context is not meant to imply that a FRET cassettehas any specificity for an Eggerthella, Prevotella, or Lactobacillustarget sequence, since it is understood that the 5′ portion of thecorresponding first probe oligonucleotide does not itself hybridize tothe respective target.

The assay for detection of Eggerthella, Prevotella, and/or Lactobacilluscan include, for each target, comparing a detection signal to apredetermined detection threshold for each target. Thresholds for eachtarget may be determined, for example, by analyzing samples from apopulation of women attending medical facilities and who have beenscored for the presence of BV using, e.g., Nugent Scores and/or theAmsel Criteria. In such embodiments, samples are assayed to determinedetection signals for each target, and a detection threshold is definedbased on the observed separation between samples from subjects who havescored positive for BV and sample from subject who have scored negativefor BV (e.g., the observed separation between Nugent positive and Nugentnegative samples). For example, in some embodiments of the methodutilizing a cleavage-based detection assay, a detection threshold isdetermined based on the initial rate of the FEN endonuclease reaction,which correlates with fluorescence signal generated from cleavage of aFRET cassette. Exemplary use of detection thresholds for determining thepresence or absence of target bacteria, based on the initial reactionrate in a cleavage-based assay, is discussed further herein in Example1.

In certain embodiments utilizing a nucleic-acid-based detection assay,the method further includes purifying the Eggerthella, Prevotella,and/or Lactobacillus target nucleic acid from other components in thesample. Such purification may include may include methods of separatingand/or concentrating organisms contained in a sample from other samplecomponents. In particular embodiments, purifying the target nucleic acidincludes capturing the target nucleic acid to specifically ornon-specifically separate the target nucleic acid from other samplecomponents. Non-specific target capture methods may involve selectiveprecipitation of nucleic acids from a substantially aqueous mixture,adherence of nucleic acids to a support that is washed to remove othersample components, or other means of physically separating nucleic acidsfrom a mixture that contains Eggerthella, Prevotella, and/orLactobacillus nucleic acid and other sample components.

In some embodiments, a target nucleic acid (e.g., a 16S rRNA targetnucleic or a gene encoding the 16S rRNA) of Eggerthella, Prevotella,and/or Lactobacillus is separated from other sample components byhybridizing the target nucleic acid to a capture probe oligomer. Thecapture probe oligomer comprises a target-hybridizing sequenceconfigured to specifically or non-specifically hybridize to a targetnucleic acid so as to form a [target nucleic acid]:[capture probe]complex that is separated from other sample components. Capture probescomprising target-hybridizing sequences suitable for non-specificcapture of target nucleic acids are described in, e.g., InternationalPCT Publication WO 2008/016988, incorporated by reference herein. In apreferred variation, the capture probe binds the [target nucleicacid]:[capture probe] complex to an immobilized probe to form a [targetnucleic acid]:[capture probe]:[immobilized probe] complex that isseparated from the sample and, optionally, washed to remove non-targetsample components (see, e.g., U.S. Pat. Nos. 6,110,678; 6,280,952; and6,534,273; each incorporated by reference herein). In such variations,the capture probe oligomer further comprises a sequence or moiety thatbinds attaches the capture probe, with its bound target sequence, to animmobilized probe attached to a solid support, thereby permitting thehybridized target nucleic acid to be separated from other samplecomponents.

In more specific embodiments, the capture probe oligomer includes a tailportion (e.g., a 3′ tail) that is not complementary to target nucleicacid but that specifically hybridizes to a sequence on the immobilizedprobe, thereby serving as the moiety allowing the target nucleic acid tobe separated from other sample components, such as previously describedin, e.g., U.S. Pat. No. 6,110,678, incorporated herein by reference. Anysequence may be used in a tail region, which is generally about 5 to 50nt long, and preferred embodiments include a substantially homopolymerictail of about 10 to 40 nt (e.g., A₁₀ to A₄₀), more preferably about 14to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence (e.g., poly-T) attached to a solidsupport, e.g., a matrix or particle.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probe oligomers that hybridize to thetarget nucleic acid under hybridizing conditions, usually at atemperature higher than the T. of the [tail sequence]:[immobilized probesequence] duplex. For embodiments comprising a capture probe tail, the[target nucleic acid]:[capture probe] complex is captured by adjustingthe hybridization conditions so that the capture probe tail hybridizesto the immobilized probe, and the entire complex on the solid support isthen separated from other sample components. The support with theattached [immobilized probe]:[capture probe]:[target nucleic acid] maybe washed one or more times to further remove other sample components.Preferred embodiments use a particulate solid support, such asparamagnetic beads, so that particles with the attached [target nucleicacid]:[capture probe]:[immobilized probe] complex may be suspended in awashing solution and retrieved from the washing solution, preferably byusing magnetic attraction. In embodiments of the method comprising theuse of an amplification-based detection assay, to limit the number ofhandling steps, a target nucleic acid may be amplified by simply mixingthe target nucleic acid in the complex on the support with amplificationoligomers and proceeding with amplification steps.

In some embodiments of a method for diagnosing BV, where detection ofEggerthella and/or Prevotella indicate BV in a subject, the methodfurther includes treating BV in the subject. Treatment regimes for BVare generally known in the art and include, for example, administrationof antibiotic drugs such as metronidazole (e.g., FLAGYL,METROGEL-VAGINAL), clindamycin (e.g., CLEOCIN, CLINDESSE), andtinidazole (e.g., TINDAMAX). In certain variations, the subject has notbeen previously diagnosed with BV. In other embodiments, the subject hasbeen previously diagnosed with BV and is undergoing treatment for BV atthe time a diagnostic method of the present disclosure is performed.Such variations are particularly useful for monitoring treatment of BVin a subject. For example, if the method indicates that BV is stillpresent in the subject, then the subject may continue treatment. In someembodiments, the same treatment regime (i.e., the same treatment thatthe subject is undergoing at the time the present diagnostic method isperformed) is re-administered to the subject. Alternatively, thecontinued presence of BV in the subject undergoing treatment mayindicate that a change in the ongoing treatment is needed, and adifferent treatment regime (e.g., a different medication, or anincreased dosage and/or frequency of a drug) is administered to thesubject.

In accordance with the present invention, detecting the presence orabsence of Eggerthella and Prevotella, or the presence or absence ofEggerthella, Prevotella, and Lactobacillus, may be performed separatelyfor each target (e.g., in separate reaction vessels, sequentially or inparallel), or performed together as a multiplex reaction system.Accordingly, in some embodiments, a method for diagnosing BV utilizes amultiplex reaction, where the reaction mix contains reagents forassaying multiple (e.g., at least two, three, four, or more) differenttarget sequences in parallel. In these cases, a reaction mix may containmultiple different target-specific oligonucleotides for performing thedetection assay. For example, in a method utilizing anamplification-based detection assay, a multiplex reaction may containmultiple sets (e.g., multiple pairs) of amplification oligomers (forexample, multiple pairs of PCR primers or multiple pairs of TMAamplification oligomers (e.g., for TMA, multiple pairs of promoterprimer and non-promoter primer, or multiple pairs of promoter providerand non-promoter primer)). In other embodiments utilizing acleavage-based detection assay, a multiplex reaction may containmultiple first probe oligonucleotides having different flaps, multipledifferent overlapping second probe oligonucleotides, and multipledifferent FRET cassettes for detecting the different flaps, once theyare cleaved. Upon cleavage of the FRET cassettes, multipledistinguishable fluorescent signals may be observed. Compounds forfluorescently labeling oligonucleotides are well-known and publicallyavailable in the art, as are the various FRET and non-FRET techniquesfor preparing and using labeled oligonucleotides containing excitationand, optionally, quenching compounds (see e.g., Dyomics GmbH, JenaGermany; Glen Research Corporation, Sterling, Va.; BiosearchTechnologies, Novato, Calif.).

Additional microbe detection assays can be similarly performed fordetermining the presence and/or relative amount of a plurality ofmicrobes implicated in BV. By way of example only, such plurality ofmicrobes can include one or more of anaerobic gram-positive cocci;Trichomonas sp.; Trichomonas vaginalis; Candida sp.; Eggerthella sp.;Bacterium from the order Clostridiales; Clostridium-like sp.; Atopobiumsp.; Atopobium vaginae; Enterobacteria; Peptostreptococcus micros;Aerococcus christensenii; Leptotrichia amnionii; Peptoniphilus sp.;Dialister sp.; Mycoplasma hominis; Sneathia sanguinegens; Anaerococcustetradius; Mobiluncus sp.; Mobiluncus hominis; Eggerthellahongkongensis; Megasphaera sp.; Leptotrichia sanguinegens and Finegoldiamagna. Assays may be performed separately or multiplexed. Thus, adiagnosis of BV can include identifying a plurality of microbes andoptionally determining their relative abundances in a sample.

In certain embodiments, the method for diagnosing BV includes thedetection of no more than ten bacterial genera associated with BV. Inother embodiments, the method includes the detection of no more thannine, no more than eight, no more than seven, no more than six, no morethan five, or nor more than four bacterial genera associated with BV. Insome variations, the method does not include detection of bacterialgenera associated with BV other than Eggerthella, Prevotella, and/orLactobacillus.

Also provided by the subject invention is a reaction mixture fordetection of an Eggerthella, Prevotella, and/or Lactobacillus targetnucleic acid. A reaction mixture in accordance with the presentinvention generally comprises an oligomer or oligomer combination asdescribed herein for detection of select species of one or more ofEggerthella, Prevotella, and Lactobacillus target nucleic acid. Thereaction mixture generally includes (i) an Eggerthella-specificoligonucleotide that specifically hybridizes to a target sequence withina target nucleic acid of an Eggerthella species characterized by thepresence of a 16S rRNA gene having the nucleobase sequence shown in SEQID NO:1, but does not specifically hybridize to a sequence within anucleic acid from other Eggerthella species, (ii) a Prevotella-specificoligonucleotide that specifically hybridizes to a target sequence withina target nucleic acid of P. amnii, P. disiens, and P. bivia, but doesnot specifically hybridize to a sequence within a nucleic acid fromother Prevotella species, and/or (iii) a Lactobacillus-specificoligonucleotide that specifically hybridizes to a target sequence withina target nucleic acid of Lactobacillus species, but does notspecifically hybridize to a sequence within a nucleic acid from L.iners. In typical variations, the reaction mixture includes at least oneEggerthella-specific oligonucleotide (e.g., at least two or threeEggerthella-specific oligonucleotides, each binding to different targetsequences) and at least one Prevotella-specific (e.g., at least two orthree Prevotella-specific oligonucleotides, each binding to differenttarget sequences); in some such variations, the reaction mixture furtherincludes at least one Lactobacillus-specific oligonucleotide (e.g., atleast two or three Lactobacillus-specific oligonucleotides, each bindingto different target sequences). The reaction mixture may further includea number of optional components such as, for example, capture probenucleic acids (e.g., a capture probe for non-specific capture of targetnucleic acids) or arrays of capture probe nucleic acids. For anamplification reaction mixture, the reaction mixture will typicallyinclude other reagents suitable for performing in vitro amplificationsuch as, e.g., buffers, salt solutions, appropriate nucleotidetriphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP),and/or enzymes (e.g., reverse transcriptase, and/or RNA polymerase), andwill typically include test sample components, in which an Eggerthella,Prevotella, and/or Lactobacillus target nucleic acid may or may not bepresent. For an cleavage-based assay reaction mixture, the reactionmixture will typically include other reagents suitable for performingformation of a cleavage structure, cleavage of the cleavage structure,and detection of the cleavage product, including, e.g., buffers, saltsolutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP,dTTP, if synthesizing a cDNA from an RNA template), and/or enzymes(e.g., a flap endonuclease and, if synthesizing a cDNA from an RNAtemplate, a reverse transcriptase), and will typically include testsample components, in which an Eggerthella, Prevotella, and/orLactobacillus target nucleic acid may or may not be present. For areaction mixture that includes a detection probe together with anamplification oligomer combination, selection of amplification oligomersand detection probe oligomers for a reaction mixture are linked by acommon target region (i.e., the reaction mixture will include a probethat binds to a sequence amplifiable by an amplification oligomercombination of the reaction mixture). For a reaction mixture thatincludes first and second overlapping probe oligonucleotides and a FRETcassette for detection via a cleavage-based assay, oligomers for areaction mixture are configured such that the FRET cassette will bind toa cleavage product produced by flap endonuclease-mediated cleavage ofthe cleavage structure formed by the first and second overlapping probeoligonucleotides, where binding of the FRET cassette to the cleavageproduct forms a secondary substrate for the flap endonuclease.

In some embodiments of a reaction mixture as above, (i) anEggerthella-specific oligonucleotide targets a sequence within anEggerthella 16S rRNA region corresponding to nucleotide positions 615 to679, (ii) an Prevotella-specific oligonucleotide targets a sequencewithin a Prevotella 16S rRNA region corresponding to nucleotidepositions 954 to 1037 of SEQ ID NO:2, and/or (iii) aLactobacillus-specific oligonucleotide targets a sequence within aLactobacillus 16S rRNA region corresponding to nucleotide positions 837to 944 of SEQ ID NO:3. In specific variations of an oligonucleotidetargeting an Eggerthella 16S rRNA region as above, theEggerthella-specific oligonucleotide includes a target-hybridizingsequence substantially corresponding to, comprising, or consisting ofthe sequence shown in SEQ ID NO:6; a target-hybridizing sequencesubstantially corresponding to, comprising, or consisting of thesequence shown in residues 11-27 of SEQ ID NO:4; or a target-hybridizingsequence substantially corresponding to, comprising, or consisting ofthe sequence shown in residues 1-20 of SEQ ID NO:5. In specificvariations of an oligonucleotide targeting a Prevotella 16S rRNA regionas above, the Prevotella-specific oligonucleotide includes atarget-hybridizing sequence substantially corresponding to, comprising,or consisting of the sequence shown in SEQ ID NO:9; a target-hybridizingsequence substantially corresponding to, comprising, or consisting ofthe sequence shown in residues 11-25 of SEQ ID NO:7; or atarget-hybridizing sequence substantially corresponding to, comprising,or consisting of the sequence shown in residues 1-24 of SEQ ID NO:8. Inspecific variations of an oligonucleotide targeting a Lactobacillus 16SrRNA region as above, the Lactobacillus-specific oligonucleotideincludes a target-hybridizing sequence substantially corresponding to,comprising, or consisting of the sequence shown in SEQ ID NO:13; atarget-hybridizing sequence substantially corresponding to, comprising,or consisting of the sequence shown in residues 11-27 of SEQ ID NO:10; atarget-hybridizing sequence substantially corresponding to, comprising,or consisting of the sequence shown in residues 1-27 of SEQ ID NO:11; ora target-hybridizing sequence substantially corresponding to,comprising, or consisting of the sequence shown in residues 1-32 of SEQID NO:12.

The invention is further illustrated by the following non-limitingexamples.

Example 1

This example describes a study combining the detection of selectEggerthella, Prevotella, and Lactobacillus species to create a test thatwas 95.6% sensitive and 97.3% specific (compared to Nugent Score).

Methods Sample Collection and Participant Demographics

The samples analyzed herein consisted of a subset of the samplescollected as part of a larger collection study. A subset of 200 samples,drawn from each of the available collection locations, were chosen forthe study described in this example. The 200 samples consisted of 99samples which were positive for BV according to the criteria used inCartwright et al. (Journal of Clinical Microbiology 51:3694-3699, 2013)and 101 which were negative.

The study population consisted of women attending medical facilities.Women must be 14 years of age or older and sign an IRB-approved waiver.Excluded from the study are premenarchal females and post-menopausalfemales. The facility collecting the samples was required to alsoprovide Nugent Scores and Amsel criteria results for each sample.Samples used for the analysis herein were collected using vaginal swabs(APTIMA® Vaginal Swab Specimen Collection kit). A total of 80 women werereported to be Caucasian, 111 African American, 2 Native American orAlaska Native, 2 Asian and for 5 subjects no race was recorded.

The sites which collected samples and reported Amsel and Nugent resultsused in this analysis were University of Alabama at Birmingham (UAB, 50samples), Louisiana State University (LSU, 50 samples), University ofWashington (UOW, 50 samples) and Women's Clinic of Lincoln, Nebr. (WCL,50 samples).

Real-Time RT-Invader® Chemistry

The ribosomal RNAs of specific bacteria were detected after conversionto cDNA using the Invader® chemistry on a Panther® system. The Invader®reaction relies on the cleavage of a specific nucleic acid structure bythe Cleavase® enzyme manufactured by Hologic and has been describedelsewhere. See, e.g., Hall et al., Proc. Natl. Acad. Sci. 97:8272-8277,1999; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999. Briefly, theCleavase® enzyme is derived from a FEN endonuclease which cleaves 5′overhangs. In the Invader detection chemistry there are two reactionswhich occur. In the primary reaction, a probe is cleaved to release a 5′fragment called the flap and in the secondary reaction, the cleaved flaphybridizes to a FRET molecule creating an overhang which allows cleavagethe 5′ end of a FRET oligo containing an attached fluorophore. The FREToligo also contains a quencher which suppresses the release offluorescence from uncleaved FRETs. When performed on the Panther®instrument for the BV assay, the primary and secondary reactions occurserially at different temperatures. In addition, fluorescence iscollected only while the secondary reaction is occurring. The net resultis an accumulation of fluorescence which directly relates to theCleavase® enzyme reaction kinetics. This allows for the estimation oftarget levels using standard enzyme kinetics or what is commonly knownas the initial rate.

The oligos for each assay included a target capture oligo, a single RTprimer, an Invader oligo, a probe oligo and a FRET oligo. The targetcapture oligo in this example was a generic oligo which hybridized to abead and indiscriminately hybridized to nucleic acid. The RT primerhybridized to the captured target and was extended by a reversetranscriptase, creating a DNA complement of the target. There was nodownstream primer so the target region was not amplified. The probeoligo hybridized to the DNA complement of the target region adjacent tothe Invader oligo, which creates the 5′ overhang in the probe. The flapof the probe was released in an Invader® reaction and the flaphybridized to the FRET, allowing the secondary Invader® reaction tocleave the FRET to release fluorescence.

The Panther instrument processed the samples as follows. The targetcapture step took place at 64° C. for 28 minutes, which was followed bya 9 minute chill and washing steps (20 minutes). The oligos, enzymes andFRETs are added and the reverse transcription step took place at 44° C.for 11 minutes and 2 seconds. This was followed by the primary reactionstep at 64° C. for 20 minutes and 34 seconds, and finally the secondaryInvader® reaction took place at 43° C. for about 53 minutes. Since themelting temperature of the flap fragment to the FRET oligo was 43° C.,the secondary reaction did not occur until the 43° C. step, which waswhen fluorescence readings were taken.

Formulations

The oligo mix consisted of 0.5 μM probe oligo, 0.25 μM Invader oligo,0.2 μM RT-primer, 0.25 μM FRET in buffer (SD PN: TN7294-108). The Enzymemix consisted of Cleavase X 700 U and MMLV 1500 U, MgCl 18 mM in buffer(SD PN: TN7294-109). All concentrations given were the final reactionconcentrations. The target capture reagent consisted of 265 mgs/mLmagnetic beads and 0.4 μM capture oligo (wobble probe; 5′-1(18T3A₃₀-3′.See e.g., WO 2008/016988 (A₉)) in APTIMA® buffer.

Oligo Sequences

The oligo sequences used in this study are included in Table 1.

TABLE 1 Oligo Sequences SEQ ID Oligo Type Target Sequence (5′ → 3′) NOProbe Eggerthella GACTAACAACgAGGCAGATGGAATTCC 4 Invader EggerthellaTGGACGACTCGAGTGCGGTAa 5 RT Primer Eggerthella GATATCTGCGCATTCCAC 6 ProbePrevotella GACCCTTATTgGCTAAGCGAAAGCA 7 Invader PrevotellaCCGCTGTTAGCACCTAGTGTTAGCa 8 RT Primer Prevotella TTGAGTTTCACCGTTGC 9Probe Lactobacillus ACAGCAAATAaGGTAGTAACTGGCCTT 10 Invader LactobacillusAGCTCTGTTGTTGGTGAAGAAGGATAGc 11 Invader LactobacillusCGTAAAGCTCTGTTGGTAGTGAAGAAAGATAGc 12 RT Primer LactobacillusTACGTATTACCGCGGCT 13 FRET* Eggerthella(F)TCT(QdT)AGCCGGTTTTCCGGCTGAGAgttgttagtc 14 FRET Prevotella(F)TCT(QdT)AGCCGGTTTTCCGGCTGAGAaataagggtc 15 FRET Lactobacillus(F)TCT(QdT)AGCCGGTTTTCCGGCTGAGAtatttgctgt 16 *For purposes of thisstudy, “F” was FAM in the FRET probe corresponding to SEQ ID NO: 14 andwas HEX in the FRET probes corresponding to SEQ ID NOs: 15 and 16;“Q” was Blackberry Quencher (BBQ) for all three FRET probes. Theselabels and label positions are exemplary only, and not limiting.

Oligo Designs Targeting Species Relevant to Bacterial Vaginosis

Oligos were designed to target only the most relevant species withineach genus for the determination of bacterial vaginosis. The oligoswhich target species in the genus Lactobacillus, did not detect the L.iners species. The oligos which targeted Eggerthella and Prevotella weresimilarly designed to target only select members of these genera. Thephylograms in FIGS. 4, 5, and 6 indicate the targeted species withineach genus and the relationship to closely related species.

In the case of Eggerthella, the focus of the design was an unculturedspecies found in the vaginal environment. See Fredricks et al., J. Clin.Microbiol. 45:3270-3276, 2007. For the Prevotella design, species whichtended to complement the BV association of the Eggerthella design werechosen. Specifically, P. amnii, P. disiens and P. bivia were targetedand other closely related species of Prevotella were not. This approachis believed to have improved sensitivity without sacrificing specificityas might be expected if all Prevotella species were included.

Fluorescence Data Collection and Analysis

The Panther instrument collected fluorescence readings for four colorsat roughly 24 second intervals during the 43° C. step in the process.The signal generated was corrected for the effects of bleed-through,detector gain and detector offset using previously obtained calibratedvalues obtained using controlled amounts of fluorescence dye and blanksAlternative corrections were explored which did not require dyecalibration and were found to be equally effective. The initial rate ofthe reaction was then calculated from the initial linear portion of thecurve. For convenience, this rate was multiplied by 1,000,000. Thisvalue is called Velocity (V).

Velocity Thresholds and Determining BV Status from Assays

Thresholds for each target were determined using the entire studypopulation. For each of the three targets, a range of roughly 0.5 logsin Velocity was the observed separation between most Nugent positive andNugent negative samples. For this study, the following threshold valueswere used. For Prevotella, the threshold was set to a log V value of2.67 (see FIG. 8); for Eggerthella, the threshold was set to a log Vvalue of 2.58 (see FIG. 9); and for Lactobacillus, the threshold was setto a log V value of 3.44 (see FIG. 10).

For each sample with a velocity value above the threshold, a value of 1was assigned; otherwise, a value of zero was assigned. This valuedetermination was performed for each target. For Eggerthella alone, thisresult was compared to either the composite result or Nugent Score. Forthe combination of select Eggerthella, Prevotella, and Lactobacillusspecies, the individual target results were combined as represented inthe flow chart shown in FIG. 7. Simply, when the velocity (V) forLactobacillus was equal to or below the threshold, samples with V valuesabove threshold for either Eggerthella or Prevotella were consideredpositive (indicative of BV). When the velocity (V) for Lactobacillus wasabove the threshold, samples with V values above threshold for bothEggerthella and Prevotella were considered positive. All other sampleswere considered to be BV negative (not indicative of BV). Thisdetermination logic can also be represented as follows: BVScore=b1+b2−g1, where b1 and b2 represent Eggerthella and Prevotella,respectively, g1 represents Lactobacillus, values assigned to b1, b2 andg1 are 0 or 1 depending on whether the V value is equal-or-below orabove the threshold, respectively; a BV score of 1 or greater is BVpositive.

Using the above logic, the assays were used to determine BV status andthis status was correlated with a composite comparator or Nugent Scorefor 200 samples.

Results

The two accepted methods of determining BV status in women are theNugent Score and Amsel Criteria. Each is often used on its own todetermine whether or not a woman should be treated for bacterialvaginosis, suggesting these are two different methods which detect thesame condition. A comparison of Amsel Criteria results to Nugent Scoredeterminations showed relatively poor performance of each test againstthe other. See Table 2. These results were similar to previousobservations. See Schwebke et al., Obstetrics & Gynecology 88:573-576,1996; Mastrobattista et al., Obstetrics & Gynecology 96:504-506, 2000.For laboratory purposes, the Nugent Score is considered to be the goldstandard. Unfortunately, a significant percentage of women will haveNugent Scores which fall into the intermediate range making diagnosisdifficult. In the study group of this example, 16% of all subjects hadNugent Scores in the intermediate range.

TABLE 2 Amesel Criteria and Nugent Score Compared Nugent Pos NegIntermediate Total Amsel Pos 61 6 8 75 Amsel Neg 30 67 28 125 Total 9173 36 200

When measured against the Nugent Score as the gold standard, the AmselCriteria were found to be 67.0% sensitive and 91.8% specific. In thiscase, 36 Nugent intermediate samples were excluded from the analysisbecause these are neither true positive nor true negative. When measuredagainst the Amsel Criteria, the Nugent Score was found to be 81.3%sensitive and 76.0% specific when Nugent Score intermediates wereconsidered to be negative. When the Nugent Score intermediates wereconsidered to be positive, the Nugent Score was 92.0% sensitive and53.6% specific when measured against the Amsel Criteria. Excluding 36Nugent Score intermediate samples yielded 91.0% sensitivity and 69.1%specificity for the Nugent Score when compared to the Amsel Criteria.

The performance of the Real-time RT-Invader assay for Eggerthella wascompared to a composite comparator which combines Amsel Criteria andNugent Score. See Table 3. For the composite comparator, a sample waspositive only if the Amsel Criteria results and the Nugent Score resultswere positive and a sample was negative only if the Amsel Criteriaresults and the Nugent Score results were negative. All other sampleswere excluded, resulting in a total of 129 samples in this analysis. Theperformance of the assay was also compared to Nugent Score alone. SeeTable 4.

TABLE 3 Real-time RT-Invader assay for Eggerthella compared toAmsel/Nugent Composite Comparator Pos Neg Total Eggerthella Positive 615 66 Eggerthella Negative 0 63 63 Total 61 68 129

TABLE 4 Real-time RT-Invader assay for Eggerthella compared to NugentScore Nugent Score Pos Neg Intermediate Total Eggerthella Positive 84 413 101 Eggerthella Negative 7 69 23 99 Total 91 73 36 200

When the Real-time RT-Invader assay for Eggerthella alone was comparedto a composite comparator, 100.0% sensitivity and 92.6% specificity werefound. When the Real-time RT-Invader assay for Eggerthella was comparedto the Nugent Score, a sensitivity of 92.3% and a specificity of 94.5%were found (with 36 Nugent Score intermediate samples excluded).

In addition to Eggerthella, the performance of assays which combine thedetection of several bacteria species were examined. Tables 5 and 6summarize the performance of an assay which targeted select Eggerthella,Prevotella, and Lactobacillus species.

TABLE 5 Real-time RT-Invader assay for Eggerthella, Prevotella &Lactobacillus compared to Amsel/Nugent Composite Comparator CompositeComparator Pos Neg Total Assay Positive 61 3 64 Assay Negative 0 65 65Total 61 68 129

TABLE 6 Real-time RT-Invader assay for Eggerthella, Prevotella &Lactobacillus compared to Nugent Score Nugent Score Pos Neg IntermediateTotal Assay Positive 87 2 15 104 Assay Negative 4 71 21 96 Total 91 7336 200

When compared to the composite comparator (Table 5), the assay performedwith 100% sensitivity and 95.6% specificity. The assay combination ofselect Eggerthella, Prevotella and Lactobacillus species produced a testwhich is 95.6% sensitive and 97.3% specific when compared to NugentScore (36 Nugent Score intermediate samples excluded).

DISCUSSION

The results for Eggerthella indicate that a test utilizing Eggerthellaalone would significantly out-perform the only FDA-approved test on themarket today when compared to the Nugent Score. Further, among thebacteria usually targeted for the diagnosis of BV, the Eggerthella assaywas found to be specific and reasonably sensitive while maintaining avery clear separation between the Nugent positive and negative samples(see FIG. 9). For contrast, high-abundance targets such as Gardnerellavaginalis tended to display a continuum of assay readings (log V)between Nugent positive and negative samples (see FIG. 11). For atargets such as Megasphaera type 1 (see FIG. 12), high specificity ispossible but at a considerable cost to sensitivity.

Previous studies have focused on combining bacterial indicators ofdysbiosis for the purpose of diagnosing bacterial vaginosis. In thestudy of this example, targets were selected and assays designed suchthat the Eggerthella and Prevotella assay have largely over-lapping andcomplementary sensitivities. In 8 out of 91 Nugent positive samples(9%), either the Eggerthella assay was positive or the Prevotella assaywas positive but not both. In 80 out of 91 Nugent positive samples(88%), both the Eggerthella and Prevotella assays were positive.

In Ravel et al. (J. Clin. Micobiol. 51:3694-3699, 2011), Prevotella wasfound 65% of the time in a population of asymptomatic women, suggestingthat targeting this genus would result in false positive results. Whendesigning the Prevotella assay of this example, only a few specificspecies of that genus were targeted. In addition, the combination ofresults for Eggerthella and Prevotella, as highly specific indicatorsfor BV, was tied with the Lactobacillus results as an indicator ofvaginal health. This approach uniquely raised both the sensitivity andspecificity of the combined assay. Both sensitivity and specificity wereimproved by combining the Eggerthella assay with assays for selectPrevotella species and select Lactobacillus species.

An underlying assumption of the assays is that Velocity relates to theabundance of the bacterial target in the sample. This was established inprevious experiments using a titration of controlled amount of bacterialtarget (example provided in FIG. 13).

The studies of this example demonstrated, inter alia, the clinicalutility of targeting select Eggerthella species for the diagnosis of BV.In this study, performance (92.3% sensitivity/93.5% specificity) whichexceeded that of the only FDA-approved test for BV on the market today.Further, unlike assays directed to some other targets, the Eggerthellaassay gave a result which clearly distinguishes between Nugent positiveand Nugent negative samples. Combining the result of the Eggerthellaassay with Prevotella and Lactobacillus resulted in a test for BV thatis highly sensitive (95.6%) and specific (97.3%) when compared to theNugent Score. Uniquely, the results of these three assays were combinedusing a logic which changes dependent on the result of the Lactobacillusassay to obtain greater sensitivity and specificity.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. A method for diagnosing Bacterial Vaginosis (BV)in a subject, the method comprising: (a) providing a sample from asubject suspected of having BV; and (b) performing a nucleic-acid-baseddetection assay for the detection of select bacterial species in each ofthe genera Eggerthella and Prevotella in the sample, wherein the assaydetects an Eggerthella species characterized by the presence of a 16SrRNA gene having a nucleobase sequence that is at least 98% identical tothe sequence shown in SEQ ID NO:1 but does not detect other Eggerthellaspecies, wherein the assay detects P. amnii, P. disiens, and P. biviabut does not detect other Prevotella species, wherein the assay targets(i) an Eggerthella 16S rRNA region corresponding to nucleotide positions615 to 679 of SEQ ID NO:1, and/or (ii) a Prevotella 16S rRNA regioncorresponding to nucleotide positions 954 to 1037 of SEQ ID NO:2, andwherein the detection of at least one of Eggerthella and Prevotellaindicates BV in the subject.
 2. The method of claim 1, wherein the assayfurther detects select Lactobacillus species in the subject but does notdetect L. iners, wherein if Lactobacillus is not detected, then thedetection of at least one of Eggerthella and Prevotella indicates BV inthe subject, and if Lactobacillus is detected, then the detection ofboth Eggerthella and Prevotella indicates BV in the subject.
 3. Themethod of claim 1, wherein the assay targets both (i) the Eggerthella16S rRNA region corresponding to nucleotide positions 615 to 679 of SEQID NO:1, and (ii) the Prevotella 16S rRNA region corresponding tonucleotide positions 954 to 1037 of SEQ ID NO:2.
 4. The method of claim1, wherein the detection of Eggerthella and Prevotella is performedusing a homogenous detection reaction.
 5. The method of claim 4, whereinthe detection of Eggerthella and Prevotella is performed in real time.6. The method of claim 1, wherein the nucleic-acid-based detection assayis a cleavage-based assay, wherein the cleavage-based assay detects anRNA target nucleic acid and utilizes a flap endonuclease that is capableof cleaving an RNA:DNA linear duplex structure, or wherein thecleavage-based assay detects a DNA target nucleic acid and utilizes aflap endonuclease that is capable of cleaving a DNA:DNA linear duplexstructure.
 7. The method of claim 6, wherein the nucleic-acid-baseddetection assay is a cleavage-based assay comprising (i) contacting thesample with (A) an Eggerthella-specific primer that specificallyhybridizes to a target sequence within SEQ ID NO:1, and (B) aPrevotella-specific primer that specifically hybridizes to a targetsequence within SEQ ID NO:2, wherein said contacting is performed underreaction conditions whereby each primer specifically hybridizes to itsrespective 16S rRNA target sequence within an Eggerthella target 16SrRNA or a Prevotella target 16S rRNA, if present; (ii) providingreactions conditions whereby the 3′ end of each hybridized primer isextended, thereby generating a single-stranded cDNA having a sequencecomplementary to a region of the Eggerthella or Prevotella target 16SrRNA, said region located 5′ to the respective primer target sequence;(iii) contacting any Eggerthella or Prevotella cDNA from step (ii) with(A) a first Eggerthella probe oligonucleotide having a 3′ portion thatspecifically hybridizes to a first target sequence within theEggerthella cDNA and a 5′ portion that does not specifically hybridizeto the Eggerthella cDNA, (B) a first Prevotella probe oligonucleotidehaving a 3′ portion that specifically hybridizes to a first targetsequence within the Prevotella cDNA and a 5′ portion that does notspecifically hybridize to the Prevotella cDNA, (C) a second Eggerthellaprobe oligonucleotide having a 5′ portion that specifically hybridizesto a second target sequence with the Eggerthella cDNA, wherein thesecond Eggerthella cDNA target sequence is located 3′ and adjacent tothe first Eggerthella cDNA target sequence, and (D) a second Prevotellaprobe oligonucleotide having a 5′ portion that specifically hybridizesto a second target sequence with the Prevotella cDNA, wherein the secondPrevotella cDNA target sequence is located 3′ and adjacent to the firstPrevotella cDNA target sequence, wherein said contacting is performedunder reaction conditions whereby if the Eggerthella cDNA is present,the first and second Eggerthella probe oligonucleotides stably hybridizeto the Eggerthella cDNA so as to form an Eggerthella linear duplexcleavage structure, and if the Prevotella cDNA is present, the first andsecond Prevotella probe oligonucleotides stably hybridize to thePrevotella cDNA so as to form a Prevotella linear duplex cleavagestructure; (iv) contacting the sample with a flap endonuclease capableof cleaving any cleavage structure from step (iii) under reactionconditions whereby if the Eggerthella cleavage structure is present,cleavage of the Eggerthella cleavage structure occurs to generate aEggerthella cleavage product comprising the 5′ portion of the firstEggerthella probe oligonucleotide, and if the Prevotella cleavagestructure is present, cleavage of the Prevotella cleavage structureoccurs to generate a Prevotella cleavage product comprising the 5′portion of the first Prevotella probe oligonucleotide; and (v) detectingthe presence or absence of the Eggerthella and Prevotella cleavageproducts.
 8. The method of claim 7, wherein the Eggerthella-specificprimer comprises the sequence shown in SEQ ID NO:6, and/or wherein thePrevotella-specific primer comprises the sequence shown in SEQ ID NO:9,and/or wherein the 3′ portion of the first Eggerthella probeoligonucleotide comprises the sequence shown in residues 11-27 of SEQ IDNO:4, and/or wherein the 3′ portion of the first Prevotella probeoligonucleotide comprises the sequence shown in residues 11-25 of SEQ IDNO:7, and/or wherein the 5′ portion of the second Eggerthella probeoligonucleotide comprises the sequence shown in residues 1-20 of SEQ IDNO:5, and/or the 5′ portion of the second Prevotella probeoligonucleotide comprises the sequence shown in residues 1-24 of SEQ IDNO:8.
 9. The method of claim 8, wherein detecting the Eggerthella andPrevotella cleavage products comprises contacting the Eggerthellacleavage product with a first FRET cassette comprising a firstfluorescent label and a first quencher, and contacting the Prevotellacleavage product with a second FRET cassette comprising a secondfluorescent label and a second quencher, preferably wherein the firstquencher and the second quencher are the same, wherein each FRETcassette hybridizes with the respective cleavage product so as to form asecond Eggerthella or Prevotella cleavage structure capable of beingcleaved by the flap endonuclease, wherein if the Eggerthella cleavageproduct is present, the first fluorescent label is released from thefirst FRET cassette comprising the first quencher, and wherein if thePrevotella cleavage product is present, the second fluorescent label isreleased from the second FRET cassette comprising the second quencher;and detecting the released first or second fluorescent label.
 10. Themethod of claim 9, (i) wherein the Eggerthella cleavage productcomprises the sequence shown in residues 1-11 of SEQ ID NO:4, whereinresidue 11 of SEQ ID NO:4 corresponds to the 3′ terminal end of saidcleavage product, and optionally wherein the first FRET cassettecomprises the sequence shown in SEQ ID NO:14, and/or (ii) wherein thePrevotella cleavage product comprises the sequence shown in residues1-11 of SEQ ID NO:7, wherein residue 11 of SEQ ID NO:7 corresponds tothe 3′ terminal end of said cleavage product and/or wherein the secondFRET cassette comprises the sequence shown in SEQ ID NO:15.
 11. Themethod of claim 9, wherein the Prevotella cleavage product comprises thesequence shown in residues 1-11 of SEQ ID NO:7, wherein residue 11 ofSEQ ID NO:7 corresponds to the 3′ terminal end of said cleavage productand/or wherein the second FRET cassette comprises the sequence shown inSEQ ID NO:15.
 12. The method of claim 2, wherein the assay targets (iii)a Lactobacillus 16S rRNA region corresponding to nucleotide positions837 to 944 of SEQ ID NO:3.
 13. The method of claim 2, wherein thedetection of Eggerthella, Prevotella, and Lactobacillus is performedusing a homogenous detection reaction.
 14. The method of claim 13,wherein the detection of Eggerthella, Prevotella, and Lactobacillus isperformed in real time.
 15. The method of claim 2, wherein thenucleic-acid-based detection assay is a cleavage-based assay, whereinthe cleavage-based assay detects an RNA target nucleic acid and utilizesa flap endonuclease that is capable of cleaving an RNA:DNA linear duplexstructure, or wherein the cleavage-based assay detects a DNA targetnucleic acid and utilizes a flap endonuclease that is capable ofcleaving a DNA:DNA linear duplex structure.
 16. The method of claim 12,wherein the nucleic-acid-based detection assay is a cleavage-based assaycomprising (i) contacting the sample with (A) an Eggerthella-specificprimer that specifically hybridizes to a target sequence within SEQ IDNO:1, (B) a Prevotella-specific primer that specifically hybridizes to atarget sequence within SEQ ID NO:2, and (C) a Lactobacillus-specificprimer that specifically hybridizes to a target sequence within SEQ IDNO:3, wherein said contacting is performed under reaction conditionswhereby each primer specifically hybridizes to its respective 16S rRNAtarget sequence within an Eggerthella target 16S rRNA, a Prevotellatarget 16S rRNA, or a Lactobacillus target 16S rRNA, if present; (ii)providing reactions conditions whereby the 3′ end of each hybridizedprimer is extended, thereby generating a single-stranded cDNA having asequence complementary to a region of the Eggerthella, Prevotella, orLactobacillus target 16S rRNA, said region located 5′ to the respectiveprimer target sequence; (iii) contacting any Eggerthella, Prevotella, orLactobacillus cDNA from step (ii) with (A) a first Eggerthella probeoligonucleotide having a 3′ portion that specifically hybridizes to afirst target sequence within the Eggerthella cDNA and a 5′ portion thatdoes not specifically hybridize to the Eggerthella cDNA, (B) a firstPrevotella probe oligonucleotide having a 3′ portion that specificallyhybridizes to a first target sequence within the Prevotella cDNA and a5′ portion that does not specifically hybridize to the Prevotella cDNA,(C) a first Lactobacillus probe oligonucleotide having a 3′ portion thatspecifically hybridizes to a first target sequence within theLactobacillus cDNA and a 5′ portion that does not specifically hybridizeto the Lactobacillus cDNA, (D) a second Eggerthella probeoligonucleotide having a 5′ portion that specifically hybridizes to asecond target sequence with the Eggerthella cDNA, wherein the secondEggerthella cDNA target sequence is located 3′ and adjacent to the firstEggerthella cDNA target sequence, (E) a second Prevotella probeoligonucleotide having a 5′ portion that specifically hybridizes to asecond target sequence with the Prevotella cDNA, wherein the secondPrevotella cDNA target sequence is located 3′ and adjacent to the firstPrevotella cDNA target sequence, and (F) a second Lactobacillus probeoligonucleotide having a 5′ portion that specifically hybridizes to asecond target sequence with the Lactobacillus cDNA, wherein the secondLactobacillus cDNA target sequence is located 3′ and adjacent to thefirst Lactobacillus cDNA target sequence, wherein said contacting isperformed under reaction conditions whereby if the Eggerthella cDNA ispresent, the first and second Eggerthella probe oligonucleotides stablyhybridize to the Eggerthella cDNA so as to form an Eggerthella linearduplex cleavage structure, if the Prevotella cDNA is present, the firstand second Prevotella probe oligonucleotides stably hybridize to thePrevotella cDNA so as to form a Prevotella linear duplex cleavagestructure, and if the Lactobacillus cDNA is present, the first andsecond Lactobacillus probe oligonucleotides stably hybridize to theLactobacillus cDNA so as to form a Lactobacillus linear duplex cleavagestructure; (iv) contacting the sample with a flap endonuclease capableof cleaving any cleavage structure from step (iii) under reactionconditions whereby if the Eggerthella cleavage structure is present,cleavage of the Eggerthella cleavage structure occurs to generate aEggerthella cleavage product comprising the 5′ portion of the firstEggerthella probe oligonucleotide, if the Prevotella cleavage structureis present, cleavage of the Prevotella cleavage structure occurs togenerate a Prevotella cleavage product comprising the 5′ portion of thefirst Prevotella probe oligonucleotide, and if the Lactobacilluscleavage structure is present, cleavage of the Lactobacillus cleavagestructure occurs to generate a Lactobacillus cleavage product comprisingthe 5′ portion of the first Lactobacillus probe oligonucleotide; and (v)detecting the presence or absence of the Eggerthella, Prevotella, andLactobacillus cleavage products.
 17. The method of claim 16, wherein theEggerthella-specific primer comprises the sequence shown in SEQ ID NO:6,and/or wherein the Prevotella-specific primer comprises the sequenceshown in SEQ ID NO:9, and/or wherein the Lactobacillus-specific primercomprises the sequence shown in SEQ ID NO:13, and/or wherein the 3′portion of the first Eggerthella probe oligonucleotide comprises thesequence shown in residues 11-27 of SEQ ID NO:4, and/or wherein the 3′portion of the first Prevotella probe oligonucleotide comprises thesequence shown in residues 11-25 of SEQ ID NO:7, and/or wherein the 3′portion of the first Lactobacillus probe oligonucleotide comprises thesequence shown in residues 11-27 of SEQ ID NO:10, and/or wherein the 5′portion of the second Eggerthella probe oligonucleotide comprises thesequence shown in residues 1-20 of SEQ ID NO:5, and/or wherein the 5′portion of the second Prevotella probe oligonucleotide comprises thesequence shown in residues 1-24 of SEQ ID NO:8, and/or wherein the 5′portion of the second Lactobacillus probe oligonucleotide comprises asequence selected from the group consisting of (1) the sequence shown inresidues 1-27 of SEQ ID NO:11 and (2) the sequence shown in residues1-32 of SEQ ID NO:12.
 18. The method of claim 17, wherein detecting theEggerthella, Prevotella, and Lactobacillus cleavage products comprisescontacting the Eggerthella cleavage product with a first FRET cassettecomprising a first fluorescent label and a first quencher, contactingthe Prevotella cleavage product with a second FRET cassette comprising asecond fluorescent label and a second quencher, and contacting theLactobacillus cleavage product with a third FRET cassette comprising athird fluorescent label and a third quencher, preferably wherein thefirst, second, and third quenchers are the same, wherein each FRETcassette hybridizes with the respective cleavage product so as to form asecond Eggerthella, Prevotella, or Lactobacillus cleavage structurecapable of being cleaved by the flap endonuclease, wherein if theEggerthella cleavage product is present, the first fluorescent label isreleased from the first FRET cassette comprising the first quencher,wherein if the Prevotella cleavage product is present, the secondfluorescent label is released from the second FRET cassette comprisingthe second quencher, and wherein if the Lactobacillus cleavage productis present, the third fluorescent label is released from the third FRETcassette comprising the third quencher; and detecting the releasedfirst, second, or third fluorescent label.
 19. The method of claim 18,(i) wherein the Eggerthella cleavage product comprises the sequenceshown in residues 1-11 of SEQ ID NO:4, wherein residue 11 of SEQ ID NO:4corresponds to the 3′ terminal end of said cleavage product andoptionally wherein the first FRET cassette comprises the sequence shownin SEQ ID NO:14, and/or (ii) wherein the Prevotella cleavage productcomprises the sequence shown in residues 1-11 of SEQ ID NO:7, whereinresidue 11 of SEQ ID NO:7 corresponds to the 3′ terminal end of saidcleavage product and optionally wherein the second FRET cassettecomprises the sequence shown in SEQ ID NO:15, and/or (iii) wherein theLactobacillus cleavage product comprises the sequence shown in residues1-11 of SEQ ID NO:10, wherein residue 11 of SEQ ID NO:10 corresponds tothe 3′ terminal end of said cleavage product, and optionally wherein thethird FRET cassette comprises the sequence shown in SEQ ID NO:16. 20.The method of claim 1, wherein the method includes the detection of nomore than ten bacterial genera associated with BV.
 21. The method ofclaim 2, wherein the method includes the detection of no more than tenbacterial genera associated with BV.